Aspergillus fumigatus has long been a focus of research, as it is the cause of the majority of Aspergillus infections. A. fumigatus is widely distributed in the environment and mainly distributed in air as conidia and is the main source of lung infection. A. fumigatus airborne counts were determined monthly during two years from the outside air environment at the University of Manchester campus and compared to total fungal airborne counts. Total fungal airborne counts were strongly seasonally associated with peak counts occurring during the summer months reaching 1,100-1400 CFU m-3and were correlated positively with mean temperature (R2=0.697). In contrast, Aspergillus fumigatus counts were not seasonally associated and gave persistent low levels of between 3-20 CFU m-3and were not correlated with mean temperature. A random selection of Manchester environmental isolates collected over one year along with clinical patient isolates and environmental isolates from the air from Dublin were analysed for genetic diversity using two combined RAPD primers. RAPD analysis revealed that the Manchester environmental isolates represented a genetically diverse population while the clinical isolates were less diverse and formed three major clusters. The Dublin isolates were the least diverse, probably due to their isolation at a single time point. When the pathogenicity of clinical and Dublin isolates were compared with a random selection of Manchester isolates in a wax moth model, as a group, clinical isolates were significantly more pathogenic than environmental isolates. Moreover, when relative pathogenicity of individual isolates was compared, clinical isolates were the most pathogenic, Dublin isolates the least pathogenic and Manchester isolates showed a range of pathogenicities suggesting that selection for the most pathogenic isolates from the environment occurs during patient infection. When the expression of secreted phospholipases in vitro during wax moth larvae of a range of isolates displaying varying degrees of pathogenicity was compared, two phospholipase C genes, AfplcA and AfplcC were strongly correlated with pathogenicity. AfplcC was by far the most highly expressed, however a ΔAfplcC knockout strain did not show attenuated virulence compared to the wild type in wax moth larvae.

Diversos estudios señalan que varias especies pertenecientes a las Bacterias Acido Lácticas (BAL) poseen propiedades probióticas y se encuentran comúnmente en los derivados lácteos. Actualmente, en la Comunidad Valenciana se comercializan quesos frescos y madurados elaborados a partir de la leche de oveja guirra, autóctona de esta comunidad e incluida dentro del catálogo de razas protegidas en España. Sin embargo, hasta el momento no se han realizado ensayos para determinar el potencial probiótico de cepas aisladas de esta leche. En esta tesis, se han aislado 131 BAL de leche de oveja Guirra. Siguiendo los criterios establecidos por la FAO, estas cepas se han identificado inicialmente con métodos fenotípicos a nivel de género y especie. Posteriormente, se ha evaluado, en condiciones in vitro, la actividad antimicrobiana de las cepas lácticas frente a patógenos implicados en enfermedades del hombre como H. pylori y Salmonella spp. En detalle, se ha estudiado el efecto de las sustancias producidas por dos cepas BAL frente a la viabilidad de H. pylori. Las cepas BAL destacadas en la actividad antimicrobiana han sido identificadas y caracterizadas por medio de técnicas moleculares, perfiles de resistencia a antibióticos, habilidad de adhesión a la mucina de cerdo y finalmente, se ha evaluado la resistencia frente a las condiciones gastrointestinales. Los resultados muestran que las propiedades probióticas se pueden atribuir a una cepa y no es posible generalizarlo a una especie o género. Algunas de las cepas lácticas de oveja y de la CECT resultaron de especial interés porque en condiciones in vitro demostraron características probióticas que deberían ser confirmadas en posteriores estudios in vivo. Cabe resaltar que algunas de estas cepas se han aislado de productos que son actualmente comercializados y por tanto las propiedades probióticas de estas cepas representarían un valor añadido para los mismos. / Amorocho Cruz, CM. (2011). Caracterización y potencial probiótico de bacterias lácticas aisladas de leche de oveja guirra [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/13830 / Palancia

The study was focused on molecular characterization of 42 clostridial strains. DNA was isolated by fenol-chloroform extraction procedure and precipitated with ethanol. After DNA isolation, PCR amplifications with specific primer sets were used for genus and species identification. Finally 19 strains were clasified as Clostridium tyrobutyricum and 3 strains were clasified as Clostridium butyricum. Presence of hydrogenase gene hydA was tested by PCR amplification using specific primer set HGf and HGr. Presence of hydrogenase gene was detected within 21 strains. (GTG)5 primer (rep-PCR) and Pr1 and Pr6 primers (RAPD) were used for differentiation of clostridial strains. Next, the cultivation of Clostridium tyrobutyricum S5 was studied under different conditions. The cultivation was carried out in liquid Reinforced Clostridial Medium (RCM) with lactose and cheese whey instead of glucose under anaerobe conditions. Growth was observed at laboratory temperature (20 to 23 °C) and at 37 °C, pH values ranging from 4.0 to 8.0 with 0.5 unit.

Bacillus popilliae and B. lentimorbus, causative agents of milky disease in Japanese beetles and related scarab larvae, have been differentiated based upon a small number of phenotypic characteristics, but they have not previously been examined at the molecular level. Thirty-four isolates of these bacteria were examined for DNA similarity. Three distinct but related similarity groups were identified; the first contained strains of B. popilliae, the second contained strains of B. lentimorbus, and the third contained two strains distinct from but related to B. popilliae. Some strains received as B. popilliae were found to be most closely related to B. lentimorbus and some received as B. lentimorbus were found to be most closely related to B. popilliae." Geographically distinct strains of B. popilliae and B. lentimorbus were analyzed using RAPD. Eight decamer primers were tested against nineteen new and seventeen isolates previously described by randomly amplified polymorphic DNA (RAPD) analysis (M. Tran). Of the new isolates, ten were found to be B. popilliae while nine isolates were more related to the B. lentimorbus species. Paraspore formation, believed to be a characteristic unique to B. popilliae, was found to occur among a subgroup of B. lentimorbus strains. Using a combination of two PCR primer pairs, the cry18Aa1 gene was detected in 31 of 35 B. popilliae isolates and in 1 of 18 B. lentimorbus isolates. When hemolymph smears were examined microscopically, a parasporal crystal was seen in three of the four B. popilliae strains where the PCR primers could not amplify the paraspore gene. The fourth strain was not tested due to the unavailability of infected hemolymph. A paraspore was also detected by microscopic examination in a subgroup of 14 B. lentimorbus strains. In combination, the primer pairs CryBp1 and CryBp2 are effective at detecting the paraspore gene in B. popilliae isolates, but not in the B. lentimorbus isolates. Growth in media supplemented with 2% NaCl was found to be less reliable in distinguishing the species than was vancomycin resistance, the latter present only in B. popilliae. The basis for vancomycin resistance in all isolates was investigated using a polymerase chain reaction assay designed to amplify the vanB gene in enterococci. An amplicon was identified and sequenced. The amplified portion of the putative ligase gene in B. popilliae had 77% and 68-69% nucleotide identity to the sequences of the vanA gene and the vanB genes, respectively. There was 75% and 69-70% identity between the deduced amino acid sequence of the putative ligase gene in B. popilliae and the deduced amino acid sequence of the vanA gene and the vanB genes, respectively. It has been determined that the vanE gene is located either on a plasmid greater than 16 kb in size or on the chromosome. The gene in B. popilliae may have had an ancestral gene in common with vancomycin resistance genes in enterococci. Bacillus sphaericus strains isolated on the basis of pathogenicity for mosquito larvae and strains isolated on the basis of a reaction with a B. sphaericus DNA homology group IIA 16S rRNA probe were analyzed for DNA similarity. All of the pathogens belonged to homology group IIA, but this group also contained nonpathogens. It appears inappropriate to designate this homology group a species based solely upon pathogenicity. / Ph. D.

Polyarthra-Rotatorien - zyklomorphe Generationen oder ein Komplex zahlreicher Arten? Elektronenmikroskopische und DNA-analytische Untersuchungen In der vorliegenden Arbeit wurde die Gattung Polyarthra (Rotatoria: Monogononta) taxonomisch untersucht. Dazu wurden licht- und rasterelektronenmikroskopische Untersuchungen sowie DNA-Analysen (RAPD-PCR) durchgeführt. Von den bisher 10 als valid geltenden Arten konnten Individuen von sieben Arten (P. euryptera, P. remata, P. major, P. dolichoptera, P. longiremis, P. vulgaris, P. luminosa) gesammelt und untersucht werden. Außerdem wurden Morphen bearbeitet, die Merkmale von verschiedenen Arten zeigten und keiner Art eindeutig zugeordnet werden konnten. Insgesamt wurden 31 verschiedene Polyarthra-Populationen aus 11 unterschiedlichen Gewässern untersucht. Ein Ziel der Arbeit bestand darin festzustellen, ob die unterschiedlichen Polyarthra-Morphen zyklomorphe einer bzw. weniger (bekannter) Arten sind oder ob sie mehrere distinkte Arten darstellen. Der Schwerpunkt der morphologischen Untersuchungen bezog sich auf den Trophus (Kauer), der bei Monogononten als artcharakteristisch gilt. Zeichnungen vom Habitus und/oder Trophus mehrerer verschiedener Morphen wurden als Originale oder aus der Literatur zusammengetragen, um einen umfangreichen Vergleich zu ermöglichen. Die DNA für die RAPD-PCR-Analysen wurde aus Einzelindividuen gewonnen, wobei die Optimierung der DNA-Isolierung im Vorfeld eigens dafür entwickelt wurde. Drei Random Primer pro Individuum konnten so angewendet werden. Sowohl morphologische als auch RAPD-Daten wurden Cluster-Analysen in Form von Parsimonie-Berechnungen unterzogen. Die RAPD-Daten wurden zusätzlich mit phänetischen Methoden berechnet. Die morphologischen und DNA-analytischen Daten lieferten danach folgende Ergebnisse: Das Auftreten von Zyklomorphose innerhalb der Polyarthren scheint eher selten zu sein. Die Validität der Arten P. euryptera, P. luminosa, P. major und P. remata konnte bestätigt werden. Nicht zweifelsfrei konnte P. vulgaris nachgewiesen werden. In Frage gestellt wird die Validität von P. dolichoptera und P. longiremis.

Both alfalfa weevil, <i>Hypera postica</i> (Gyllenhal), (Coleoptera: Curculionidae), and potato leafhopper, <i>Empoasca fabae</i> (Harris), (Homoptera: Cicadellidae), remain key pests of alfalfa in Virginia. Commercial varieties of potato leafhopper-resistant (or glandular-haired) alfalfa were released in the mid-1990s, but the impact of alfalfa weevil on these varieties has not been well documented. In 1999, two large-scale field experiments were initiated to compare the performance of a glandular-haired alfalfa variety against a standard, non-glandular-haired variety under both alfalfa weevil and potato leafhopper pest pressures in the southwestern and Piedmont regions of Virginia over a 3 year period. Results indicated that alfalfa weevil must be managed in potato leafhopper-resistant alfalfa to limit crop loss. Surprisingly, similar densities of potato leafhoppers were found in both the glandular-haired and standard varieties. Both varieties frequently had similar yields and forage quality. In general, the glandular-haired variety did not outperform the standard variety. Results also indicated that insecticide application did not always provide the expected benefits of higher yields and forage quality, despite reducing pest densities for 2-3 weeks after application. These data suggest that the economic thresholds for one or both of these pests in Virginia may require adjustment. The potential impact of glandular-haired alfalfa on the natural enemies of alfalfa weevil was examined as well. <i>Bathyplectes anurus</i> (Thompson) (Hymenoptera: Ichneumonidae) was the dominant parasitoid attacking weevil larvae at both locations. Parasitization of weevil larvae by <i>Bathyplectes</i> spp. did not appear to be adversely affected by the presence of glandular trichomes on the potato leafhopper-resistant variety. Glandular trichomes had little impact on the infection of weevil larvae by the fungus <i>Zoophthora phytonomi</i> as well, but this was not unexpected. The genetic variation of <i>B. anurus</i> was surveyed at both study sites using RAPD-PCR to establish or eliminate the possibility that differences in parasitization levels between the Piedmont and southwestern regions could be attributed to the presence of different parasitoid strains. Most of the detected phenotypic variation was attributed to within population variation, with very little variation occurring between the two populations. However, the between population variation was statistically significant in 2000, but not in 2001. / Ph. D.

Zwei Populus-Pfropfchimären (MA und AI), die aus P. x canadensis ‘Marilandica’ (M), P. maximowizcii x P. trichocarpa ‘Androscoggin’ (A) and P. nigra L. ’Italica’ (I) aufgebaut sind, wurden für Untersuchungen zur Laub- und Blütenblattentwicklung genutzt. In MA bildet M die äußere Lage (L1) und ihr Derivat, die Epidermis, während die inneren Lagen (L2, L3 etc.) von A gebildet werden. Bei AI stammt die L1 von A und L2, L3 etc. werden von I gebildet. Die genotypisch andersartige Epidermis bedingt bei Periklinal-Chimären morphologische Effekte wie zum Beispiel einen Fruchtknoten in einigen MA-Blüten. Morphologische Besonderheiten verschiedener Gewebe sowohl von M und A als auch von MA wurden verglichen, um festzustellen, wie sie durch die Gewebetransplantation verändert oder beeinflusst wurden und, um mögliche Genotyp- Interaktionen oder -Wechselwirkungen in einem Gewebe ausfindig zu machen. Für die Genotypidentifizierung in verschiedenen Organen wurde die RAPD-PCR getestet. Einer von 20 getesteten 10mer Zufallsprimern (GGAGTGGACA) ermöglichte bei der Verwendung von DNA aus Blattmaterial die Erzeugung verschiedener Bandenmuster für M und A. Bei der Verwendung von MA-Blattmaterial zeigte sich eine Kombination der Muster von M und A, sodass ein Chimärennachweis für das MA-Blattmaterial erbracht wurde. Für ein übertragbares System wurde die spezifische PCR getestet. Unter Verwendung spezifischer Primer für die 16s-rDNA zeigten die PCR-Produkte einheitliche Banden und nach anschließender Sequenzierung eine weitgehende Übereinstimmung der phylogenetischen Verwandtschaft von I, M und A. Weiterhin wurden die kernkodierten rDNA Bereiche ITS 1 und ITS 2 zwischen 18S und 25S getestet. Für I, M und A konnten jeweils zwei Banden von unterschiedlicher Größe und Sequenz ermittelt werden, die vermutlich auf funktionierende rDNA aber auch auf Pseudogene (beschnitten) in niedriger Kopienzahl hinweisen. Die ITS-Regionen von I, M und A wurden charakterisiert, um einen Einblick in die Struktur und Phylogenie der Salicacaee-Familie zu erhalten. Aus den Sequenzunterschieden konnten für I und A spezifische Primerpaare abgeleitet werden, die für die Identifizierung von I und A in AI und MA verwendet werden können. Mittels A-Marker konnte nachgewiesen werden, dass Fruchtknoten aus MA-Blüten neben M-Gewebe auch den A-Genotyp enthalten. / Two Populus graft chimeras (MA and AI) produced of P. x canadensis ‘Marilandica’ (M), P. maximowizcii x P. trichocarpa ‘Androscoggin’ (A) and P. nigra L. ’Italica’ (I) were used for investigations of leaf and flower development. In MA the exogenous layer (L1) forms the epidermis and is derived from M while inner layers (L2, L3 etc.) descend from A whereas in AI L1 is formed by A while L2, L3 etc. descend from I. The exogenous epidermis of the periclinal chimeras imposes morphological effects such as an extra female sex in some of the MA flowers. The morphological characteristics of different plant tissues of parents and chimera were compared to determine how they were modified or altered by the tissue transplantation and possibly identify co-existing or interacting genotypes in one tissue. RAPD-PCR was tested for its usefulness to amplify polymorphic fingerprints including donor specific DNA fragments. One random 10mer primer (GGAGTGGACA) out of 20 tested revealed the amplification of patterns including donor specific DNA bands using extracts from leaf tissues of the M and A parents that were combined using extract from leaf tissue of the MA chimera. This indicates that the leaves of the MA chimera are formed by tissues of M and A. However, the inherent disadvantage of RAPD-PCR is the reproducibility of PCR product generation. Therefore the discriminative potential of the ITS region located between the rRNA genes was investigated. The application of specific 16S ribosomal DNA (rDNA) primers for amplification and sequencing of PCR products revealed a closely phylogenetic relationship between I, M and A. Consequently the ITS1 and ITS2 of nuclear rDNA between 18S and 25S were used. The amplified fragments were purified, cloned in E. coli and sequenced. Analyses of multiple clones demonstrated extensive paralogy within and between I, M and A ITS operons. For each parent were at least two rDNA operons as well as multiple paralogous sequences within operons identified. The use of PCR and sequence analyses showed that one of the operons encodes a putative expressed (functional) rDNA whereas the second encodes a pseudogen (truncated) in low copy number. We also characterized the ITS regions of I, M and A to gain insights into structure and phylogeny of the Salicacaee family. Based on sequence divergence primers were designed for A and I and used for the identification of A in MA carpels.

A rubelose é uma doença que atinge galhos e ramos, e é causada pelo fungo Erythricium salmonicolor, o qual infecta várias espécies vegetais, tais como citros, seringueira, e macieira. Esta doença vem chamando a atenção dos citricultores devido à redução de até 10% da produção de citros, a qual é preocupante para o Brasil, o maior produtor de laranja do mundo. Entretanto, a diversidade do fungo E. salmonicolor em cultivares brasileiras ainda não foi avaliada. Desta maneira, este trabalho teve como objetivos: i) avaliar a variabilidade genética, por meio de RAPD, de isolados do fungo E. salmonicolor provenientes de diferentes regiões citrícolas de São Paulo e Minas Gerais, ii) avaliar a compatibilidade vegetativa e fusão de hifas deste fungo e iii) selecionar bactérias endofíticas com potencial para o controle deste fungo fitopatogênico. Após a análise por RAPD, foram observados 6 grupos distintos (A, B, C, D, E, F), os quais não apresentaram correlação com o local de origem e espécie hospedeira. No teste de compatibilidade vegetativa houve encontro de hifas em todos os cruzamentos e 84% destes apresentaram fusão entre as hifas. Foi verificada compatibilidade entre linhagens, embora não tenha sido observada correlação com os haplótipos. No teste de antagonismo, 8 isolados bacterianos inibiram E. salmonicolor. Entretanto, foi observada diferença na interação entre as bactérias e diferentes isolados de E. salmonicolor, visto que bactérias diferentes inibiram os dois genótipos do fungo, revelando a variabilidade genética entre estas linhagens que pertencem a diferentes haplótipos. Os resultados observados neste trabalho indicam a importância de futuros estudos sobre a fase sexual de E. salmonicolor, uma vez que a anastomose de hifas precede a formação de heterocário, responsável pelos processos de recombinações sexuais e parassexuais, que geram variabilidade genética em fungos filamentosos. / The fungus Erythricium salmonicolor is the causal agent of pink disease, which infects branches of many host plants, such as citrus, rubber, and apple. This disease may be a serious problem in Brazil, since it can reduce the citrus production up to 10%. Brazil is the major world citrus producer, therefore this problem is alarming. The genetic diversity of E. salmonicolor from Brazilian plants has not been evaluated, so the aims of this study were i) to evaluate the genetic diversity by RAPD of E. salmonicolor isolates from São Paulo and Minas Gerais; ii) to evaluate the vegetative compatibility and hyphal fusion of this fungus; and iii) to select endophytic bacteria able to inhibit the E. salmonicolor growth. RAPD analysis showed at least 6 distinct haplotypes (A, B, C, D, E, F), which did not have correlation with the isolation site and host plant. Also, vegetative compatibility tests showed that 84% of crosses resulted in hyphal fusion, but this compatibility was not related to the RAPD haplotypes. Eight endophytic bacteria were selected against E. salmonicolor, which could be used for biological control of this pathogen. However it was observed different types of interaction among endophytic bacteria and E. salmonicolor strains, since these bacteria inihibited differentially two fungi isolates. It reveals the genetic variability between these fungi isolates that belongs to different haplotypes These results show the importance of future studies concerning the sexual phase of E. salmonicolor, since the genetic variability seems to be high and this hyphal fusion, which precede the formation of heterokaryon (sexual and parassexual reproduction), could be responsible for the variability in this filamentous fungus.

Pseudomonas fluorescens EPS62e es va seleccionar com a agent de biocontrol del foc bacterià per la seva eficàcia en el control de Erwinia amylovora. En aquest treball es van desenvolupar mètodes de traçabilitat que van permetre la seva detecció específica i quantificació. Mitjançant les tècniques RAPD i U-PCR es van obtenir fragments d'amplificació diferencial per EPS62e que es van seqüenciar i caracteritzar com marcadors SCAR per dissenyar una PCR en temps real. La PCR a temps real es va utilitzar simultàniament amb mètodes microbiològics per estudiar l'adaptabilitat epifítica de EPS62e en pomera i perera. L'ús combinat de mètodes microbiològics i moleculars va permetre la identificació de tres estats fisiològics de EPS62e: la colonització activa, l'entrada en un estat de viable però no cultivable, i la mort cel·lular. Aquest treball mostra que EPS62e està ben adaptada a la colonització de flors a camp, encoratjant la seva utilització dins d'una estratègia de control biològic contra el foc bacterià. / Pseudomonas fluorescens EPS62e was selected as a reliable biological control agent of fire blight for its high efficacy controlling Erwinia amylovora infections. In the present work, monitoring methods which allowed EPS62e specific detection and quantification were developed. RAPD and U-PCR fingerprints were used to obtain differential amplified fragments from EPS62e that were sequence characterized as SCAR markers. A real-time PCR was developed on the basis of the strain-specific SCAR markers, and was used simultaneously with microbiological methods to study the environmental fate of EPS62e in apple and pear orchards. The combined use of both microbiological and molecular methods permitted the identification of three physiological states for EPS62e, which consisted of active colonization, survival and entry into a viable but nonculturable state, and cell death. The present work shows that EPS62e is well adapted for blossom colonisation in the field, and encourages its utilisation in a fire blight.

Molecular markers were used in an attempt to determine the phylogenetic relationships of hexaploid wheats within Triticum aestivum L. and to identify wheat cultivars. Random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), gliadin protein and cytological analyses were used to assess phylogenetic relationships among five morphological groups of hexaploid wheat, namely, macha, common wheat, spelta, vavilovii and semi-wild wheat (SWW). RAPD and gliadin data were analysed using the NTSYS-pc computer program to generate Jaccard genetic similarity coefficients. Coefficients of genetic similarity in the cytological study were calculated based on the number of chiasmata in hybrids. Dendrograms were constructed based on these coefficients. The dendrogram based on RAPD analysis grouped 15 accessions into five distinct clusters which were in agreement with the morphology-based classification. The results indicated that common wheat was closely related to vavilovii. Spelta was less related to the common and vavilovii wheat cluster. SWW was distantly related to common wheat. Macha was the least related to the previous clusters. These results were consistent with those based on cytological analysis. The results of gliadin analysis were not completely consistent with those based on RAPD and cytological analyses. RFLP data showed that it was difficult to determine phylogenetic relationships among the five groups of hexaploid wheat based on variation in the intergenic spacer region of the 18-25S rRNA unit. Polymerase chain reaction analysis of the 5S rRNA unit and the internal transcribed spacer of the 18-25S rRNA unit did not show any polymorphism among and within the five groups of hexaploid wheat. Twelve mis-classified Triticum accessions were found in macha and vavilovii wheat collections and investigated using RAPD and cytological analyses. A dendrogram based on RAPD analysis classified the 12 accessions into either T. monococcum, T. turgidum spp. dicoccum or T. timopheevii. The results were in agreement with cytogenetic data and morphological observations. The genetic diversity of spelta and macha wheat was also investigated using RAPD analysis and the results were generally consistent with geographic origins. Macha wheat germplasm was found slightly more diverse than spelta wheat although macha has a restricted geographic origin. In addition, duplicate accessions of macha and spelta were identified based on RAPD analysis. In the study of wheat cultivar identification and pedigree assessment, 29 cultivars were investigated using RAPD analysis. Cultivar specific markers were found, and at least eight cultivars could be identified using these specific markers. Cultivar relationships based on genetic similarity values were consistent with knownpedigrees. The study demonstrated that RAPD analysis can be used for estimating the phylogenetic relationships among the five groups of hexaploid wheat, reclassifying misclassified wheat germplasm, surveying the genetic diversity of spelta and macha wheat and identifying common wheat cultivars and duplicated accessions in wheat germplasm collections.