PCR-based detection of sunflower white blister rust (<Emphasis Type="Italic">Pustula helianthicola</Emphasis> C. Rost &; Thines) in soil samples and asymptomatic host tissue

Sequencing of partial cox2 (part of the mitochondrial cytochrome-c-oxydase (COX) gene) was performed with samples from the oomycete genus Pustula, the white blister rusts of Asteraceae and related families. Sequence comparison uncovered several single nucleotide polymorphisms (SNPs) between P. spinulosa and host specific strains of Pustula isolated from Senecio vulgaris, Tragopogon pratensis and cultivated sunflower, Helianthus annuus. Based on these differences, specific primers were designed for PCR-based detection of white blister rust strains pathogenic to sunflower. The specificity of the primers was confirmed by cross testing with DNA from various oomycetes occurring in the same locality. The limit of detection for DNA of P. helianthicola was 10 pg. This allowed detection with DNA from single sporangia and single oospores. The PCR-based experiments allowed detection of the presence of sunflower white blister rust in soil samples from fields on which infected plants had been cultivated several months before. Moreover, the molecular tools were successfully applied to trace the pathogen in asymptomatic tissue of infected plants, demonstrating the systemic nature of Pustula on sunflower.     

Rotenone and rotenoids in cubè resins, formulations, and residues on olives

Rotenone and rotenoids (deguelin, beta-rotenolone (12a beta-hydroxyrotenone), tephrosin (12a beta-hydroxydeguelin), 12a alpha-hydroxyrotenone, and dehydrorotenone) were determined in cubè resins and formulations. Cubè resins from Lonchocarpus contain large quantities of deguelin (ca. 21.2%) and smaller quantities of tephrosin (ca. 3.5%) and beta-rotenolone (ca. 3.0%). The composition of commercial formulations may present very different rotenoid contents depending on the extracts used to prepare them. Because these rotenoids also present insecticide activity, the efficacy of these formulations may be very different. The storage stability and photodegradation of some rotenone formulations were studied. Rotenone and rotenoids are very sensitive to solar radiation, which degrades them rapidly, with half-lives in the order of a few tens of minutes. Some formulations show greater disappearance rates than that of cubè resin, indicating that not much attention has been paid to protecting the active ingredients from photodegradation in the formulation. A study on the residues on olives was also carried out to assess not only the rotenone content, but also that of the main rotenoids. At harvest, the residues of deguelin, tephrosin, and beta-rotenolone were 0.10, 0.06, and 0.10 mg/kg, respectively, very similar to rotenone (0.08 mg/kg), and though a few data indicate similar acute toxicity values for deguelin, only rotenone is taken into consideration in the legal determination of the residue.     

Genome evolution following host jumps in the Irish potato famine pathogen lineage

Many plant pathogens, including those in the lineage of the Irish potato famine organism Phytophthora infestans, evolve by host jumps followed by specialization. However, how host jumps affect genome evolution remains largely unknown. To determine the patterns of sequence variation in the P. infestans lineage, we resequenced six genomes of four sister species. This revealed uneven evolutionary rates across genomes with genes in repeat-rich regions showing higher rates of structural polymorphisms and positive selection. These loci are enriched in genes induced in planta, implicating host adaptation in genome evolution. Unexpectedly, genes involved in epigenetic processes formed another class of rapidly evolving residents of the gene-sparse regions. These results demonstrate that dynamic repeat-rich genome compartments underpin accelerated gene evolution following host jumps in this pathogen lineage.     

Environmental correlates of two macro-decapods distribution in Central Italy: Multi-dimensional ecological knowledge as a tool for conservation of endangered species

Using a multi-dimensional ecological design, this study aimed first to analyse whether local environmental conditions can account for the spatial segregation of two Italian native decapods, the crayfish Austropotamobius italicus and the river crab Potamon fluviatile, in Central Italy freshwater ecosystems. Second, we aimed to analyse which environmental variables were more closely associated with the presence/absence of the two decapods in specific sites within their distribution area. Following a factorial design, a total of 32 sites were selected in two neighbouring geographic areas, one occupied by crayfish and one by crabs. Within each distribution area, eight streams where the decapod was present and eight where it was not present were selected. At each site, macro-invertebrate community composition and 16 abiotic variables were recorded and analysed with multi-variate methods. Variations in physical (minimum and maximum temperatures), chemical (calcium, oxygen, nitrate and nitrite) and geomorphological (substrate composition) parameters explained spatial segregation of P. fluviatile and A. italicus in the study area. The occurrence of crayfish reflected variations of chemistry (such as pH, calcium, nitrate and nitrite concentrations), temperature, water depth and substrate composition. On the contrary, the presence of the river crab, within its occurrence zone, was not associated to any biotic and abiotic parameters and was probably affected by anthropogenic pressure and uncontrolled harvesting. These findings provide fundamental ecological data for the maintenance of the two decapod natural populations as well as for the selection of areas and streams adequate for their reintroduction.     

Sea turtle strandings reveal high anthropogenic mortality in Italian waters

• 1. Spatio‐temporal distribution and anthropogenic mortality factors were investigated in loggerhead turtles (Caretta caretta) found stranded or floating in the waters around Italy. A total of 5938 records for the period 1980–2008 were analysed concerning loggerhead turtles measuring from 3.8 to 97 cm curved carapace length (mean: 48.3 cm).
• 2. Results highlighted the following conservation issues: (i) in the study area, anthropogenic mortality is higher than natural mortality; (ii) interaction with fisheries is by far the most important anthropogenic mortality factor; (iii) longlines are an important mortality factor in the southern areas; (iv) trawlers are the cause of high numbers of dead strandings in the north Adriatic; (v) entanglement in ghost‐gear or in other anthropogenic debris affects high numbers of turtles; and (vi) boat strikes are an important source of mortality in most areas but mostly in the warm seasons.
• 3. Results also indicate that: (vii) the north Adriatic is the area with the highest turtle density; and (viii) the south Adriatic and to a lesser extent the surrounding areas of the north Adriatic and the Ionian, are important developmental areas for loggerhead turtles in the first years of life.
• 4. Italy is in the centre of the Mediterranean Sea and borders major foraging areas for the loggerhead turtles in the region, and these results confirm previous concerns about the level of anthropogenic mortality in Italian waters. Copyright © 2010 John Wiley & Sons, Ltd.

     

Portable surface plasmon resonance immunosensor for the detection of fluoroquinolone antibiotic residues in milk

An inexpensive and portable surface plasmon resonance (SPR) sensor, SPReeta Evaluation Kit SPR3, has been used to develop a biosensor for the determination of fluoroquinolone antibiotics (FQs) and to demonstrate its performance analyzing FQ residues in milk samples. The SPReeta three-channel gold chips were activated with a mixed self-assembled monolayer (m-SAM) and functionalized with a FQ haptenized protein. Binding of the antibody produced a concentration-dependent increase of the SPR signal as a result of the change in the refraction index. Similarly, the presence of the FQ produced a dose-dependent decrease of the response, which allowed a good limit of detection (LOD) to be obtained (1.0 ± 0.4 μg L(-1) for enrofloxacin in buffer). The response was reproducible in all three channels, on different injections and days, and also between chips. Milk samples could be analyzed after a simple sample treatment involving fat removal by centrifugation and dilution with water. Under these conditions calibration curves were obtained showing that FQ residues can be analyzed in milk samples with an IC(50) value of 26.4 ± 7.2 μg L(-1) and a LOD of 2.0 ± 0.2 μg L(-1) (for enrofloxacin), far below the European Union regulations for this antibiotic family in this matrix. Finally, the paper also demonstrates that the biosensor is able to selectively detect the presence of FQs in milk samples, even in the presence of other antibiotics. Enrofloxacin, ciprofloxacin, and norfloxacin residues were detected in blind samples supplied by Nestle? Co.     

Comparison of different media for the isolation of small ruminant strains of Mycobacterium paratuberculosis

Two different egg based media, with and without the incorporation of sodium pyruvate, were used to isolate M. paratuberculosis from sheep, goat and cattle samples obtained at our laboratory for two years. Both media were adequate for bovine material, with a slightly improved isolation rate for Herrold's egg yolk medium incorporating sodium pyruvate; however, most of the small ruminant strains grew only on L?wenstein-Jensen medium without sodium pyruvate. Those results point out the need to use different media when working with small ruminant samples and provide further evidence for the existence of different varieties of M. paratuberculosis causing paratuberculosis in cattle and in small ruminants.