Massimo Labra

Resolution of viable and membrane-compromised bacteria in freshwater and marine waters based on analytical flow cytometry and nucleic acid double staining.

ABSTRACT The membrane integrity of a cell is a well-accepted criterion for characterizing viable (active or inactive) cells and distinguishing them from damaged and membrane-compromised cells. This information is of major importance in studies of the function of microbial assemblages in natural environments, in order to assign bulk activities measured by various methods to the very active cells that are effectively responsible for the observations. To achieve this task for bacteria in freshwater and marine waters, we propose a nucleic acid double-staining assay based on analytical flow cytometry, which allows us to distinguish viable from damaged and membrane-compromised bacteria and to sort out noise and detritus. This method is derived from the work of S. Barbesti et al. (Cytometry 40:214–218, 2000) which was conducted on cultured bacteria. The principle of this approach is to use simultaneously a permeant (SYBR Green; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable. In the present study, this approach has been adapted to bacteria in freshwater and marine waters of the Mediterranean region. It is fast and easy to use and shows that a large fraction of bacteria with low DNA content can be composed of viable cells. Admittedly, limitations stem from the unknown behavior of unidentified species present in natural environments which may depart from the established permeability properties with respect to the fluorescing dyes.

Evidence of a secondary grapevine domestication centre detected by SSR analysis

Abstract. The origin of the grapevine was investigated with archaeobotanical, cultural and historical data. A primary domestication centre was located in the Near East region but there is no agreement on the existence or role of secondary domestication centres. In this work, PCR-based microsatellite analysis has been applied to study the origin of some Italian cultivated grapevines from in situ direct domestication of the wild autoctonous grapevine. Three different Italian locations in Grosseto, Cosenza and Nuoro were identified for this study, and domesticated grapevine as well as wild local accessions growing in these location, were analysed by SSR markers. Cluster analysis performed on Cosenza and Grosseto samples showed a high value of genetic distance between domesticated and wild accessions. On the contrary two cultivars (Bovale Murru and Bovale Muristellu) recovered in Nuoro (in the Sardinia island) were very close to some wild varieties. This suggests that the latter two cultivars may have originated from wild grapevines and consequently that in this location a secondary grapevine domestication event occurred. Six Lambrusco varieties were also included in this analysis as ancient putative ancestors of the cultivated grapevines. The molecular analysis excluded this hypothesis and suggest Lambrusco as an independent Vitis taxon.

Two and three-color fluorescence flow cytometric analysis of immunoidentified viable bacteria

BACKGROUND Traditional culture methods well established in the past and still in use are not able to detect the environmental microorganisms that exist in a viable but not culturable state. A number of different fluorescence-based assays have been developed over the past decade to detect and identify viable bacteria in the environment. METHODS We have developed a simple and rapid method for measuring the number and viability of immunolabeled bacteria by means of a two/three color fluorescence flow cytometric analysis. After washing, cultured bacteria in suspension were labeled with a rabbit polyclonal antibody recognizing the wall lipopolysaccharide complex. A secondary biotinylated anti-rabbit polyclonal antibody was added allowing the cells to be labeled with the streptavidin R-phycoerythrin-Cyanine 5 (RPE-Cy5) fluorochrome. Before flow cytometric analysis, bacterial suspensions were stained with SYBR Green I and propidium iodide which stain all of the cells and the non viable ones, respectively. RESULTS AND CONCLUSIONS With the appropriate filter sets of both Bryte-HS (Bio-Rad, Hercules, CA) and FACScan (Becton Dickinson, San Jose, CA) flow cytometers, the measurement of separated green (SYBR Green I), orange-red (propidium iodide), and far red (RPE-Cy5) fluorescence was possible, allowing the enumeration of viable immunodetected bacteria. The entire protocol is completed in less than 3 h, offering numerous possibilities for rapid and precise analyses in sanitary, industrial, and environmental microbiology.

Identification of poisonous plants by DNA barcoding approach

The plant exposures are one of the most frequent poisonings reported to poison control centres. The diagnosis of intoxicated patients is usually based on the morphological analysis of ingested plant portions; this procedure requires experience in systematic botany, because the plant identification is based on few evident traits. The objective of this research is to test DNA barcoding approach as a new universal tool to identify toxic plants univocally and rapidly. Five DNA barcode regions were evaluated: three cpDNA sequences (trnH-psbA, rpoB and matK) and two nuclear regions (At103 and sqd1). The performance of these markers was evaluated in three plant groups: (1) a large collection of angiosperms containing different toxic substances, (2) congeneric species showing different degrees of toxicity and (3) congeneric edible and poisonous plants. Based on assessments of PCR, sequence quality and resolution power in species discrimination, we recommend the combination of plastidial and nuclear markers to identify toxic plants. Concerning plastidial markers, matK and trnH-psbA showed consistent genetic variability. However, in agreement with CBOL Plant Working Group, we selected matK as the best marker, because trnH-psbA showed some problems in sequences sizes and alignments. As a final and relevant observation, we also propose the combination of matK with a nuclear marker such as At103 to distinguish toxic hybrids form parental species. In conclusion, our data support the claim that DNA barcoding is a powerful tool for poisonous plant identifications.

AFLP analysis as biomarker of exposure to organic and inorganic genotoxic substances in plants

In recent years several plant species have been in use as bioindicators and several tests have been developed to evaluate the toxicity of environmental pollutants in vegetal organisms. In the present paper Arabidopsis thaliana (L.) Heynh. (ecotype Wassilewskija) was used as bioindicators of two genotoxic substances: potassium dichromate and dihydrophenanthrene. Inhibition of seed germination was observed with both pollutants. AFLP analysis (i) indicated that both substances are genotoxic, (ii) showed that dihydrophenanthrene induces DNA changes in different target sequences than potassium dichromate, (iii) quantified the genotoxic effect using cluster analysis by comparing DNA from treated plants with that of control plants. On the basis of these considerations we suggest that AFLP method is a powerful tool for measuring qualitative and quantitative genotoxic activity due to environmental pollutants. AFLP method can be applied to a wide range of bioindicator organisms and may become a universal methodology to identify target genes for specific genotoxic agents. This could open up possibilities for designing specifically targeted assays and new approaches to risk assessment.

A DNA barcoding approach to identify plant species in multiflower honey

The purpose of this study was to test the ability of DNA barcoding to identify the plant origins of processed honey. Four multifloral honeys produced at different sites in a floristically rich area in the northern Italian Alps were examined by using the rbcL and trnH-psbA plastid regions as barcode markers. An extensive reference database of barcode sequences was generated for the local flora to determine the taxonomic composition of honey. Thirty-nine plant species were identified in the four honey samples, each of which originated from a mix of common plants belonging to Castanea, Quercus, Fagus and several herbaceous taxa. Interestingly, at least one endemic plant was found in all four honey samples, providing a clear signature for the geographic identity of these products. DNA of the toxic plant Atropa belladonna was detected in one sample, illustrating the usefulness of DNA barcoding for evaluating the safety of honey.

A DNA Barcoding Approach to Characterize Pollen Collected by Honeybees

In the present study, we investigated DNA barcoding effectiveness to characterize honeybee pollen pellets, a food supplement largely used for human nutrition due to its therapeutic properties. We collected pollen pellets using modified beehives placed in three zones within an alpine protected area (Grigna Settentrionale Regional Park, Italy). A DNA barcoding reference database, including rbcL and trnH-psbA sequences from 693 plant species (104 sequenced in this study) was assembled. The database was used to identify pollen collected from the hives. Fifty-two plant species were identified at the molecular level. Results suggested rbcL alone could not distinguish among congeneric plants; however, psbA-trnH identified most of the pollen samples at the species level. Substantial variability in pollen composition was observed between the highest elevation locality (Alpe Moconodeno), characterized by arid grasslands and a rocky substrate, and the other two sites (Cornisella and Ortanella) at lower altitudes. Pollen from Ortanella and Cornisella showed the presence of typical deciduous forest species; however in samples collected at Ortanella, pollen of the invasive Lonicera japonica, and the ornamental Pelargonium x hortorum were observed. Our results indicated pollen composition was largely influenced by floristic local biodiversity, plant phenology, and the presence of alien flowering species. Therefore, pollen molecular characterization based on DNA barcoding might serve useful to beekeepers in obtaining honeybee products with specific nutritional or therapeutic characteristics desired by food market demands.

DNA Barcoding as an Effective Tool in Improving a Digital Plant Identification System: A Case Study for the Area of Mt. Valerio, Trieste (NE Italy)

Background Identification keys are decision trees which require the observation of one or more morphological characters of an organism at each step of the process. While modern digital keys can overcome several constraints of classical paper-printed keys, their performance is not error-free. Moreover, identification cannot be always achieved when a specimen lacks some morphological features (i.e. because of season, incomplete development or miss-collecting). DNA barcoding was proven to have great potential in plant identification, while it can be ineffective with some closely related taxa, in which the relatively brief evolutionary distance did not produce differences in the core-barcode sequences. Methodology/Principal Findings In this paper, we investigated how the DNA barcoding can support the modern digital approaches to the identification of organisms, using as a case study a local flora, that of Mt. Valerio, a small hill near the centre of Trieste (NE Italy). The core barcode markers (plastidial rbcL and matK), plus the additional trnH-psbA region, were used to identify vascular plants specimens. The usefulness of DNA barcoding data in enhancing the performance of a digital identification key was tested on three independent simulated scenarios. Conclusions/Significance Our results show that the core barcode markers univocally identify most species of our local flora (96%). The trnH-psbA data improve the discriminating power of DNA barcoding among closely related plant taxa. In the multiparametric digital key, DNA barcoding data improves the identification success rate; in our simulation, DNA data overcame the absence of some morphological features, reaching a correct identification for 100% of the species. FRIDA, the software used to generate the digital key, has the potential to combine different data sources: we propose to use this feature to include molecular data as well, creating an integrated identification system for plant biodiversity surveys.

Genomic stability in Arabidopsis thaliana transgenic plants obtained by floral dip

The occurrence of DNA modification is an undesired phenomenon accompanying plant cell transformation. The event has been correlated with the stress imposed by the presently utilised transformation procedures, all depending on plant differentiation from in vitro cell culture, but other causes have not been excluded. In this work, transgenic Arabidopsis thaliana plants have been produced by an approach that does not require cell dedifferentiation, being based on in planta Agrobacterium-mediated gene transfer by flower infiltration, which is followed by recovery and selection of transgenic progeny. Genomic DNA changes in transgenic and control plants have been investigated by AFLP and RAMP analysis. Results show no statistically relevant genomic modifications in transgenic plants, as compared with control untreated plants. Variations were observed in callus-derived A. thaliana plants, thus supporting the conclusion that somaclonal variation is essentially correlated with the stress imposed by the in vitro cell culture, rather than with the integration of a foreign gene.

Genetic relationships in Opuntia Mill. genus (Cactaceae) detected by molecular marker

The Opuntia genus includes over 181 species comprising, on the basis of morphological traits, a total of 29 series [The Cactaceae (1919)]. Starting from this classification, several authors have investigated the Opuntia genus taxonomy but the large morphological variation within different species, suggests that phenotypical characteristics will not serve to produce a stable classification. In this work chloroplastic simple sequence repeat (cpSSR) and amplified fragment length polymorphism (AFLP) were used to evaluate the usefulness of molecular markers in Opuntia species characterization and to study the relationships among different species. Results show that the combination of cpSSR and AFLP markers provide a quantitative estimation of genetic relationships among several Opuntia species. Both molecular analyses reveal a genetic similarity among species of series 20 and 21 [The Cactaceae (1919)] as suggested also by morphological traits. Particular attention was focused on the genetic relationship between Opuntia ficus-indica and Opuntia megacantha: individuals from different populations of the two species were analyzed with both molecular markers. A common genetic constitution of O. ficus-indica and O. megacantha was detected. On the basis of molecular data, morphological traits and biogeographical distribution, we suggest that O. ficus-indica should be considered as a domesticated form of O. megacantha. Our results suggest the importance of a revision of Opuntia genus classification using several tools: molecular, morphological and biogeographical analysis.