Elena Vendramin

Soil humic compounds and microbial communities in six spruce forests as function of parent material, slope aspect and stand age

The influences on soil chemical and microbial properties of parent material, north south aspect and time measured as stand age were investigated in six spruce (Picea abies (L.) Karst.) forests located in the alpine range of Northern Italy. Soil samples from A horizons were analysed for humic substances and in parallel Amplified Ribosomal DNA Restriction Analysis (ARDRA) community profiles and microbial biomass carbon and nitrogen content were determined. Chemical data were analyzed by canonical discriminant analysis while the ARDRA fingerprints were ordered in clusters using image analysis software. The geologic parent material was the most determining factor and the aspect-dependent microclimate features also played a distinct role in defining both soil chemistry and microbial community composition; in contrast the composition of the deeper humus layers (OH, A) was stable and similar within a spruce forest cycle time. Most important variables in the construction of the discriminating models resulted soil pH, Dissolved Organic Carbon content and Dissolved Organic Matter phenolic compounds. Bacterial communities appeared to be shaped first and foremost by the substratum, secondly by mountain slope orientation, and thirdly by forest stage, thus confirming the CDA model.

Identification of Enterococcus mundtii as a pathogenic agent involved in the “flacherie” disease in Bombyx mori L. larvae reared on artificial diet

Enterococcus mundtii was shown to be directly correlated with flacherie disease of the silkworm larvae reared on artificial diet supplemented with chloramphenicol. Its identification was carried out by means of light and electron microscopy and nucleotide sequencing of 16S gene. The bacterium is capable of rapidly multiplying in the silkworm gut and of invading other body tissues, as demonstrated by deliberate infection of germfree larvae and by subsequent TEM observations. E. mundtii can endure alkaline pH of the silkworm gut and it has been proved to adapt in vitro to commonly applied doses of chloramphenicol, whose use can further contribute to reduce competition by other bacteria in Bombyx mori alimentary canal. The modality of transmission of the infection to the larvae was among the objectives of the present research. Since contamination of the progeny by mother moths can be avoided through routine egg shell disinfection, a trans-ovarian vertical transmission can be ruled out. On the other hand the bacterium was for the first time identified on mulberry leaves, and therefore artificial diet based on leaf powder could be a source of infection. We showed that while microwaved diet could contain live E. mundtii cells, the autoclaved diet is safe in this respect. Being E. mundtii also part of the human-associated microbiota, and since B. mori is totally domestic species, a possible role of man in its epidemiology can be postulated.

Bacterial diversity of a wooded riparian strip soil specifically designed for enhancing the denitrification process

This research is part of a project aimed at verifying the potential of a specifically assessed wooded riparian zone in removing the excess of combined nitrogen from the Zero River so as to reduce nutrient inputs into the Venice Lagoon. Among the specific objectives of the project, there was the determination of change in the composition of the microbial populations of soil of the wooded riparian strip. The composition of the bacterial communities collected at different depths inside and outside the riparian strip was determined by combined approaches involving cultivation (CFU), microscopic approaches (CTC test), and DNA-based techniques (ARDRA and DGGE). The size of the living population was the same inside and outside the experimental strip, with a minor percentage of culturable bacteria. Higher numbers of metabolically active bacteria and higher bacterial diversity were detected in the internal soil, with deeper soil layers showing reduced diversity, thus indicating that soil management within the riparian strip effectively supports the viability of bacterial communities. Total operational taxonomic units (OTUs) and percentage of single OTUs were also found to be always higher in the internal soil samples for all soil layers, with the percentage of Firmicutes increasing and Actinobacteria decreasing with depth. The increasing soil organic carbon inputs due to the contribution of the growing plants were found to support bacterial diversity in all soil layers. DNA-based analysis also indicated a clear effect of the applied treatments on soil bacterial diversity and a well-defined separation of the bacterial communities related to the different soil layers of the riparian strip.

Aspects of marker/reporter stability and selectivity in soil microbiology

Based on several experiences of microbial release using genetically modified Rhizobium leguminosarum, we have highlighted a number of aspects related to the suitability of introduced markers such as resistance to mercury and β-galactosidase activity, the latter serving the function of high-expression level reporter gene obtained by the introduction of a synthetic promoter conferring strong inducible expression in Gram-negative bacteria. In vitro expression and in vivo performances of the chosen examples have been followed in model strains comparing gene dosage and expression levels. The technical possibility of unambiguously monitoring the marked GMM has been evaluated in medium- and long-term experiments carried out both in microcosms and soil, also including the presence of the plant symbiotic host. Marker stability, regardless the nature of the gene, was shown to be dependent on the location of the genetic modification and on its degree of gene expression regulation. Reporter strength was found to be an advantage allowing the distinction of marker-bearing bacterial while negatively affecting their genetic stability. Plasmid-borne regulated reporters were found to be stable up to the stages of rhizosphere colonization, but were more critically selected against upon symbiotic host invasion.

Fungi associated with the southern Eurasian orchid Spiranthes spiralis (L.) Chevall

The hitherto unknown relationships between the European orchid Spiranthes spiralis (L.) Chevall and its internally associated fungi were explored by a combined approach involving microscopy-based investigations at a morpho-histological level as well as by molecular analyses of the identity of the eukaryotic endophytes present in the root tissue of the plant. We found that this orchid which is currently reported to have a vulnerable status in northern Italy, can host and interact with at least nine types of fungi. Some of these fungi show similarity to mycorrhizal genera found in orchids such as the Ceratobasidium-Rhizoctonia group. Other fungi found are from the genera Davidiella (Ascomycota), Leptosphaeria (Ascomycota), Alternaria (Ascomycota), and Malassezia (Basidiomycota), some of which until have not previously been reported to have an endophytic relationship with plants. The repeated occurrence of often pathogenic fungi such as Fusarium oxysporum, Bionectria ochroleuca, and Alternaria sp., within healthy specimens of this orchid suggests a tempered interaction with species that are sometimes deleterious to non-orchid plants. The fact is reminiscent of the symbiotic compromise established by orchids with fungi of the rhizoctonia group.

Taxonomical analysis of the suspended bacterial fraction in the dromedary rumen fluid

An analysis of the dominant microbial taxa present in suspension within the rumen fluid from slaughtered one-humped camel (Camelus dromedarius ) in Algeria was carried out using cultureindependent molecular techniques. The rumen fluid of freshly eviscerated animals was sampled by a syringe and filtered through 0.22 μm filters in sterile conditions. Lyophilized filters were subsequently used as starting material for bacterial lysis and total DNA extraction procedures using DNA purification kits and suitably adapted protocols. The gene corresponding to the small subunit of ribosomal RNA (16S rDNA) was PCR-amplified from the bulk of DNA using eubacterial primers, and the pool of amplicons was ligated to plasmids and cloned in Escherichia coli, generating a clone bank of several hundred individuals representative of the rumen bacterial community. A preliminary analysis of 86 clones, sorted by amplified ribosomal DNA restriction analysis (ARDRA), and sequenced by Applied Biosystems automated sequencing using fluorescent terminators yielded the following results. The most abundant amplicon belonged to the Pseudomonas genus encompassing over 65% of the clones. Pseudomonas lutea appeared the most frequent homology hit in a BLAST GenBank comparison. The remaining flora featured taxa include (in order of deceasing abundance): Synechococcus sp., Moraxella osloensis, Sphingomonas sp., Diaphorobacter nitroreducens, Acinetobacter sp., Ruminococcus albus, Propionibacterium acnes and Comamonas sp . The data constitute the baseline for a comparison of the results with those that will be obtained by further metagenomic approaches to compare the fluid associated bacterial community with those attached to the solid particulate fraction. Key words : Camelus dromedarius, dromedary camel, rumen fluid, rumen bacteria.

Environmental Impact of Genetically Modified Rhizobium Leguminosarum bv. viciae

Experiments aimed at assessing the effects following the field release of genetically modified microorganisms (GMO) were carried out. Derivatives of different strains of Rhizobium leguminosarum bv. viciae were constructed carrying environmentally-neutral reporter genes or resistance determinants to non-antibiotic compounds, and released in soil in the presence of pea host plants. Genes were chosen that would not confer selective advantage to the GMOs in the environment of choice, in order to evaluate any net impact of a genetic modification per se. Two strains were used as background and tagged with the same modifications, the first was isolated from a location distant from the site of the release, the second represented the dominant pea-nodulating strain within the indigenous populations of the site. These differences of origin played a major role in the ecological outcome of the symbiosis, in that the foreign strain, behaved extremely poorly, both in persistance and in infection, being severely outcompeted by the natural rhizobia, while the autochtonous derivative maintained the competitive dominant attitude of its parental version, occupying the majority of the host nodules. However, monitoring the effects of both releases by viable plate counts of different microbial groups including aerobic bacteria, fungi, actynomycetes, fluorescent pseudomonads, other rhizobia and some representative soil activities, did not reveal any perturbations, confirming that a genetic modification per se, does not inherently produce an impact even when carried by a species that succesfully and extensively colonizes its habitat