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.

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.

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.

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.

Retrotransposon and gene activation in wheat in response to mycotoxigenic and non-mycotoxigenic-associated Fusarium stress.

Despite inhibition of protein synthesis being its mode of action, the trichothecene mycotoxin deoxynivalenol (DON) induced accumulation of transcripts encoding translation elongation factor 1alpha (EF-1alpha), class III plant peroxidase (POX), structure specific recognition protein, basic leucine zipper protein transcription factor (bZIP), retrotransposon-like homologs and genes of unknown function in the roots of wheat cultivars CM82036 and Remus. Fusarium head blight (FHB) studies using Fusarium graminearum and its trichothecene-minus (Tri5 ( - )) mutant derivative and adult plant DON tests showed that these transcripts were responsive to both mycotoxigenic- and non-mycotoxigenic-associated Fusarium stress. In tests using the parents 'CM82036', 'Remus' and 14 double-haploid progeny that segregated for quantitative trait locus (QTL) Fhb1 on chromosome 3BS (syn. Qfhs.ndsu-3BS) (from 'CM82036' that confers DON tolerance), bZIP expression was significantly more DON-up-regulated in lines that inherited this QTL. Basal accumulation of the bZIP transcript in spikelets treated with Tween20 (control), DON and in DON-relative to Tween20-treated spikelets was negatively correlated with DON-induced bleaching above (but not below) the treated spikelets (AUDPC(DON)) (r = -0.41, -0.75 and -0.72, respectively; P < or = 0.010). bZIP-specific PCR analysis of 'Chinese spring' and its 3BS deletion derivatives indicated that bZIP is located in chromosomal region(s) other than 3BS. These results, and the fact that a homologous cold-regulated wheat bZIP (wLIP19) maps to group 1 chromosomes suggests that wheat bZIP may participate in defence response cascades associated with Fhb1 and that there is a cross-talk between biotic and abiotic stress signalling pathways.

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).

Blood-meal analysis for the identification of reservoir hosts of tick-borne pathogens in Ireland.

The results of analysis of blood-meal remnants in unfed nymphs, despite relatively low detection levels (49.4%, n = 322), support the conclusion from an earlier study that small rodents are relatively unimportant as reservoir hosts of B. burgdorferi s.l. in this particular area, and suggest that songbirds (Passeriformes) are the most significant hosts in this respect. Tick (Ixodes ricinus) abundance was greater in the present study, but the overall Borrelia burgdorferi s.l.-infection prevalence of nymphal ticks was the same (12.2%), and the relative proportions of the various Borrelia burgdorferi s.l. genospecies were similar. B. garinii and B. valaisiana were the most frequent, B. burgdorferi s.s the least frequent, and B. afzelii of intermediate frequency. An unusually high proportion of nymphs (39%) with multiple infections of different B. burgdorferi genospecies was detected, and Borrelia spp. related to relapsing-fever spirochetes were detected in Ireland for the first time. The results of the present study contribute to the validation of blood-meal analysis as a means of determining the host origin of certain pathogens in unfed questing ticks, and raise some questions concerning the extent of B. burgdorferi s.l. host specificity.

Detection and identification of pathogens and host DNA in unfed host-seeking Ixodes ricinus L. (Acari: Ixodidae).

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.