Upward movement of Verticillium dahliae from soil to olive plants detected by qPCR.

Olive trees play an important role in cultural, ecological, environmental and social fields, constituting in large part the Mediterranean landscape. In Tuscany, an important economic activity is based on olive. Unfortunately, the Verticillium wilt affects this species and causes vascular disease. In the present study, a real-time quantitative PCR approach has been used to detect and quantify Verticillium dahliae in soil and in olive tree tissues both in micropropagated and in seedling olives. The minimum amounts of V. dahliae DNA sequences detected in soil were 11.4 fg which is equivalent to less than one fungal haploid genome. In micropropagated olive the pathogen was detected in the leaves after 43 days, showing a vertical upward movement of the fungus from the culture medium to stem and leaves. A similar fungal behaviour was observed in inoculated olive stem where after 15 days the fungal DNA was detected from symptomless stem tissue above 8 cm the inoculation site. The described molecular approach is expected to provide a more sensitive and less time-consuming alternative detection method for V. dahliae than plating assay procedures, which were traditionally proposed as an early diagnosis method for Verticillium wilt to farmers and tree nursery growers.

Experimental discrimination and molecular characterization of the extracellular soil DNA fraction.

We experimentally discriminated and qualitatively-quantitatively characterized the extracellular fraction of a forest soil DNA pool. We sequentially extracted and classified the components of extracellular DNA by its strength of interaction with soil colloids as: (1) extractable in water, free in the extracellular soil environment or adsorbed on soil colloids; and as (2) extractable in alkaline buffer after previous extraction in water, bound on soil colloids. The comparative molecular analysis (fluorometer, gel electrophoresis, genetic fingerprinting) of directly and sequentially extracted extracellular DNA revealed quantitative and qualitative differences, also in terms of genetic information about microbial communities. The sequential extraction of extracellular DNA revealed differences in molecular weight, indicating a relationship between DNA fragment length and strength of interaction with soil colloids. The sequential extraction was also suitable to assess the presence of tightly bound DNA, providing information about the DNA-colloid interactions naturally occurring in the soil environment.

Long-term effects of aided phytostabilisation of trace elements on microbial biomass and activity, enzyme activities, and composition of microbial community in the Jales contaminated mine spoils.

We studied the effectiveness of remediation on microbial endpoints, namely microbial biomass and activity, microbial and plant species richness, of an As-contaminated mine spoil, amended with compost (C) alone and in combination with beringite (B) or zerovalent iron grit (Z), to increase organic matter content and reduce trace elements mobility, and to allow Holcus lanatus and Pinus pinaster growth. Untreated spoil showed the lowest microbial biomass and activity and hydrolase activities, and H. lanatus as sole plant species, whereas the presented aided phytostabilisation option, especially CBZ treatment, significantly increased microbial biomass and activity and allowed colonisation by several plant species, comparable to those of an uncontaminated sandy soil. Microbial species richness was only increased in spoils amended with C alone. No clear correlation occurred between trace element mobility and microbial parameters and plant species richness. Our results indicate that the choice of indicators of soil remediation practices is a bottleneck.

Persistence of transgenic and not transgenic extracellular DNA in soil and bacterial transformation.

The study of the fate of transgenic and not transgenic extracellular DNA in soil is of extreme relevance because the soil extracellular DNA pool represents a genetic reservoir that could be utilized as a source of food by any heterotrophic microorganism or genetic information by recipient eukaryotic and prokaryotic cells. Several data have clearly evidenced that extracellular DNA could persist in soil for long time maintaining a sufficient integrity of the molecule. Recent microcosm studies under laboratory conditions have evidenced that extracellular DNA molecule could be leached or raised up by capillarity. The persistence and movement of extracellular DNA molecule in soil suggest that the genetic information of extracellular DNA could be taken up by microorganisms temporarily and spatially separated. Several authors have studied the persistence and transformation efficiency of the extracellular DNA in soil demonstrating that there is a sharp discrepancy between its biological efficiency and its persistence; fragments of target DNA were detected after a long time in soil but no transformations were determined probably because the genetic information originally present in the complete DNA molecule could be lost by degradation. It is also important to underline that the frequency of gene transfer in soil is markedly limited by the few number of bacteria able to develop competence and that this physiological state is reached only under certain conditions. Furthermore the dilution of the transgene in the soil extracellular DNA pool drastically decreases chances for the uptake of the transgene. Anyway the importance of transformation in evolutionary terms, represents a valid reason to continue the investigation on the fate of extracellular DNA in soil.