Damian Egan

Detection and Identification of Pathogens and Host DNA in Unfed Host-Seeking Ixodes ricinus L. (Acari: Ixodidae)

Abstract In this study, we have developed molecular methods for the identification of reservoir hosts of sylvatic tick-borne zoonoses. The methods are based on the analysis of the blood meal remnant in the tick gut and include detection of pathogens and identification of the host origin of the blood meal. For host identification, a universal primer pair was used to amplify part of the vertebrate 18S rRNA gene followed by reverse line blot hybridization using subgroup-specific probes. Analyses of DNA from whole blood of vertebrates identified the correct subgroup of a broad range of vertebrate species (e.g., Ruminantia, Leporidea, Canidae, Murinae, Arvicolinae, Insectivora, Galliformes, Passeriformes) using probes based on the 18S rDNA sequences. Host DNA in the remnants of larval blood meals was detected in the gut of Ixodes ricinus nymphs maintained under natural conditions up to 9 mo after molting. For pathogen identification, a multiplex polymerase chain reaction was used that targeted parts of the 18S rRNA gene of piroplasm protozoa, the 16S rRNA gene of bacteria, and the intergenic spacer of the Borrelia burgdorferi genospecies complex. The utility of both methods was demonstrated under laboratory conditions by detecting Babesia microti (Franca) and gerbil DNA in 3-mo-old I. ricinus nymphs that had fed on B. microti-infected gerbils as larvae, and under field conditions by analyzing unfed ticks that were collected in a forest. The field study showed that the majority of ticks had fed on ruminants or birds and few on rodents, which is in accord with our knowledge of the fauna in this forest. Few pathogens were detected but the discovery of Borrelia valaisiana and B. burgdorferi s.s. in ticks that had fed on deer and Borrelia afzelii in a tick that had fed on a bird raises questions about the mode of transmission of these spirochetes and possibly about their host specificity.

Gut wall bacteria of earthworms: a natural selection process

Earthworms and microorganisms are interdependent and their interactions regulate the biogeochemistry of terrestrial soils. Investigating earthworm–microorganism interactions, we tested the hypothesis that differences in burrowing and feeding habits of anecic and endogeic earthworms are reflected by the existence of ecological group-specific gut wall bacterial communities. Bacterial community was detected using automated ribosomal intergenic spacer analysis of 16S and 23S genes and ribotype data was used to assess diversity and community composition. Using soil and earthworm samples collected from adjacent wheat–barley and grass–clover fields, we found that the anecic Lumbricus terrestris and L. friendi, the endogeic Aporrectodea caliginosa and A. longa (classically defined as anecic, but now known to possess endogeic characteristics) contain ecological group-specific gut wall-associated bacterial communities. The abundance of specific gut wall-associated bacteria (identified by sequence analysis of ribotype bands), including Proteobacteria, Firmicutes and an actinobacterium, was ecological group dependent. A microcosm study, conducted using A. caliginosa and L. terrestris and five different feeding regimes, indicated that food resource can cause shifts in gut wall-associated bacterial community, but the magnitude of these shifts did not obscure the delineation between ecological group specificity. Using A. caliginosa and A. longa samples collected in six different arable fields, we deduced that, within an ecological group, habitat was a more important determinant of gut wall-associated bacterial community composition than was host species. Hence, we conclude that the selection of bacteria associated with the gut wall of earthworms is a natural selection process and the strongest determinant of this process is in the order ecological group>habitat>species.

Biological control of fusarium seedling blight disease of wheat and barley.

ABSTRACT Fusarium fungi, including F. culmorum, cause seedling blight, foot rot, and head blight diseases of cereals, resulting in yield loss. In a screen for potential disease control organisms and agents, Pseudomonas fluorescens strains MKB 100 and MKB 249, P. frederiksbergensis strain 202, Pseudomonas sp. strain MKB 158, and chitosan all significantly reduced the extent of both wheat coleoptile growth retardation and wheat and barley seedling blight caused by F. culmorum (by 53 to 91%). Trichodiene synthase is a Fusarium enzyme necessary for trichothecene mycotoxin biosynthesis; expression of the gene encoding this enzyme in wheat was 33% lower in stem base tissue coinoculated with Pseudomonas sp. strain MKB 158 and F. culmorum than in wheat treated with bacterial culture medium and F. culmorum. When wheat and barley were grown in soil amended with either chitosan, P. fluorescens strain MKB 249, Pseudomonas sp. strain MKB 158, or culture filtrates of these bacteria, the level of disease symptoms on F. culmorum-inoculated stem base tissue (at 12 days post- F. culmorum inoculation) was >/=31% less than the level on F. culmorum-inoculated plants grown in culture medium-amended soil. It seems likely that at least part of the biocontrol activity of these bacteria and chitosan may be due to the induction of systemic disease resistance in host plants. Also, in coinoculation studies, Pseudomonas sp. strain MKB 158 induced the expression of a wheat class III plant peroxidase gene (a pathogenesis-related gene).

Identification of benzimidazole resistance in Cladobotryum dendroides using a PCR-based method

Cladobotryum dendroides, causal agent of cobweb disease of Agaricus bisporus, has become increasingly resistant to methylbenzimidazole carbamate (MBC) fungicides following the extensive use of MBC in cultivated mushroom production in Ireland. Of 38 isolates of C. dendroides obtained from Irish mushroom units, 34 were resistant to carbendazim. Primers based on conserved regions of the β-tubulin gene were used to amplify and sequence a portion of the β-tubulin gene in C. dendroides. A point mutation was detected at codon 50 in isolates resistant to benzimidazole fungicides, causing an amino acid substitution from tyrosine to cysteine. Species-specific PCR primers were designed to amplify the region of the β-tubulin gene containing this substitution. The point mutation removed an Acc I restriction site in the β-tubulin gene sequence of resistant isolates. Digestion of the PCR product with Acc I thus provides a rapid diagnostic test to differentiate sensitive and resistant isolates of this fungus. EMBL accession number: Y12256.

Components of the gene network associated with genotype-dependent response of wheat to the Fusarium mycotoxin deoxynivalenol

The Fusarium mycotoxin deoxynivalenol (DON) facilitates fungal spread within wheat tissue and the development of Fusarium head blight disease. The ability of wheat spikelets to resist DON-induced bleaching is genotype-dependent. In wheat cultivar (cv.) CM82036 DON resistance is associated with a quantitative trait locus, Fhb1, located on the short arm of chromosome 3B. Gene expression profiling (microarray and real-time RT-PCR analyses) of DON-treated spikelets of progeny derived from a cross between cv. CM82036 and the DON-susceptible cv. Remus discriminated ten toxin-responsive transcripts associated with the inheritance of DON resistance and Fhb1. These genes do not exclusively map to Fhb1. Based on the putative function of the ten Fhb1-associated transcripts, we discuss how cascades involving classical metabolite biotransformation and sequestration processes, alleviation of oxidative stress and promotion of cell survival might contribute to the host response and defence against DON.

Detection and quantification ofSpongospora subterranea f. sp.subterranea by PCR in host tissue and naturally infested soils

A polymerase chain reaction (PCR) assay using primers SsF and SsR designed from the internal transcribed spacer (ITS) regions ofSpongospora subterranea f. sp.subterranea was developed for the specific identification and quantification ofS. subterranea. These primers amplified a 434 bp product from DNA ofS. subterranea spore balls, but not from DNA of healthy potato, common scab tuber, and taxonomically related plasmodiophorids. This PCR assay was successfully used for the detection ofS. subterranea in naturally infected symptomatic and asymptomatic potato tubers.Spongospora subterranea in other infected symptomless host plants was detected by PCR. The PCR assay was modified with improved soil DNA extraction methods to detectS. subterranea in soil. The assay was sensitive, and one spore ball per gram of soil could be detected. Following the design of a heterologous competitor DNA template from the sequence of λDNA, a competitive PCR assay for the quantification ofS. subterranea in soil was developed and provided accurate quantification in the range of 1 to 104 spore balls per 0.25 g of soil. In a preliminary survey of naturally infested field soil samples, spore ball concentrations were estimated to vary from ca 0 to 3600 spore balls per 0.25-g soil sample by this competitive PCR assay. The spore ball levels were compared with the powdery scab disease incidence of potatoes in these fields, and a correlationship between spore ball levels and subsequent disease incidence was found. The PCR assays developed in this investigation can be routinely used to detect and quantifyS. subterranea in diseased plant tissue, asymptomatic plant tissue, and infested soil.ResumenLa prueba de reacción en cadena de la polimerasa (PCR) utilizando los “primers” SsF y SsR, diseñados a partir de las regiones transmitidas internamente del espaciador (ITS) deS. subterránea f. sp.subterranea, ha sido desarrollada para la identificación específica y cuantificación deSpongospora subterranea. Estos “primers” amplificaron el producto 434 bp del DNA de las masas de esporas deS. subterranea pero no del DNA de papa sana, o de tubérculos con sarna común y plasmodiophoridos taxonómicamente relacionados. Esta prueba de PCR ha sido utilizada exitosamente para la detección deS. subterranea en tubérculos naturalmente infectados, sintomáticos y asintomáticos. También se ha detectado a través del PCR,S. subterranea en otros huéspedes infectados que no presentaban síntomas. La prueba PCR ha sido modificada con métodos mejorados de extracción de DNA del suelo para detectarS. subterranea. La prueba es tan sensible que pude detectar una espora por gramo de suelo. Después del diseño de una muestra competitiva heteróloga de DNA de la secuencia de λDNA se desarrolló una prueba competitiva de PCR para la cuantificaciónS. subterranea en el suelo, la misma que proporcionó una cuantificación segura en el rango de 1 a 104 masas de esporas por 0.25 gramos de suelo. En un estudio preliminar de muestras de suelo infestado naturalmente, se estimó que la concentración varió de c. 0 a 3600 masas de esporas por 0.25 gramos de muestra de suelo por medio de esta prueba de PCR. El nivel de masas de esporas se comparó con la incidencia de sarna polvorienta en estos campos de papa y se encontró una correlación entre los niveles de masas de esporas e incidencia de la enfermedad en una siguiente campaña. Las pruebas de PCR desarrolladas en esta investigación pueden ser rutinariamente utilizadas para detectar y cuantificarS. subterranea en el tejido de plantas enfermas, tejido de plantas asintomáticas y suelo infestado.

Field preservation and DNA extraction methods for intestinal microbial diversity analysis in earthworms

We assessed the effect of DNA extraction and sample preservation methods on the DNA yield and quality obtained from earthworm (Aporrectodea caliginosa Savigny) gut samples and on the results obtained by bacterial and fungal automated ribosomal intergenic spacer analysis (ARISA) of DNA extracts. Methods based on a hexadecyltrimethylammonium bromide dithiotreitol (CTAB-DTT) extraction buffer yielded more favourable results than those based on a sodium dodecyl sulphate (SDS) buffer. For both of these buffers, incorporation of a bead-beating during the lysis step increased the ARISA-derived bacterial ribotype numbers and diversity estimates, as determined for gut wall samples (P<0.01). Although spectrophotometric analysis indicated that DNA extracted by the CTAB-DTT and SDS-based methods were of comparable quality (P> or =0.05), the former method yielded >1.5 times more DNA from both gut contents and gut walls of earthworms than the latter method (both incorporating the bead beating step) (P<0.01). ARISA analysis detected more reproducible ribotypes and more microbial diversity in DNA extracted by the CTAB-DTT- as compared to the SDS-based method (P<0.01). Significant difference between bacterial communities of gut contents and gut walls were detected within DNA extracted by the CTAB-DTT (but not by the SDS-based) method (Global R=0.76, P<0.001, analysis of similarity). Using the CTAB-DTT-based method, we showed that earthworm preservation in ethanol yielded higher quality DNA from gut contents than preservation in either chloroform or liquid N, as determined by spectrophotometry, PCR inhibition analysis and bacterial and fungal ARISA (P<0.05). Bacterial or fungal communities in the gut contents of fresh and ethanol-preserved earthworms were more similar and were significantly different from those of earthworms preserved in chloroform or liquid N (Global R=0.79 and 0.83 for bacteria and fungi, respectively; P<0.001, analysis of similarity). We propose that ethanol preservation and the CTAB-DTT-based DNA extraction method described herein are also suitable for the analysis of gut-associated microbiota in other soil and sediment feeding invertebrates.

Morphological and molecular characterisation of Penicillium roqueforti and P. paneum isolated from baled grass silage

The morphological and molecular features of Penicillium roqueforti and P. paneum isolated from baled grass silage were characterised. A total of 315 isolates were investigated, comprising 237 P. roqueforti and 78 P. paneum isolates randomly selected from more than 900 Penicillium colonies cultured from bales. The macromorphological features of both species broadly agreed with the literature, but the micromorphological features differed in some respects. When observed using SEM, P. roqueforti and P. paneum had finely roughened conidia, and conidiophores, phialides and conidia of P. paneum were each larger than those of P. roqueforti. Based on the phylogenetic analysis of partial sequences of beta-tubulin and acetyl co-enzyme A (CoA) synthetase genes, P. roqueforti and P. paneum isolates were found to be monophyletic species.

An eDNA assay for Irish Petromyzon marinus and Salmo trutta and field validation in running water.

This pilot study presents an environmental DNA (eDNA) assay for sea lamprey Petromyzon marinus and brown trout Salmo trutta, two species of economic and conservation importance in the Republic of Ireland. The results demonstrate the effectiveness of eDNA for assessing presence of low-abundance taxa (here, P. marinus) for environmental managers, and they highlight the potential for assessing relative abundance of rare or invasive freshwater species.