In vitro inhibition of Citrobacter freundii, a red-leg syndrome associated pathogen in raniculture, by indigenous Lactococcus lactis CRL 1584

Red-leg syndrome (RLS) is one of the main infectious diseases that cause economic losses in Lithobates catesbeianus hatcheries, Citrobacter freundii being an etiological agent. Treatment or prevention with therapeutics or chemicals results in modifications of the indigenous microbiota, development of antibiotic resistance, presence of their residues in food and enhancement of production costs. Thus, probiotics could be used as an alternative therapy. Lactic acid bacteria are part of the indigenous microbiota of healthy frogs and can prevent pathogen colonization by different mechanisms, including the production of antagonistic substances. In this work, the evaluation and characterization of the inhibition of C. freundii CFb by Lactococcus lactis subsp. lactis CRL 1584, a potentially probiotic candidate, were carried out. This strain produced lactic acid, H(2)O(2) and bacteriocin in static and shaken conditions and inhibited pathogen growth in associative cultures, with an earlier inhibition under agitated conditions. The elimination of each of the antimicrobial metabolites partially abolished the inhibition of the pathogen, suggesting that the inhibitory effect could be attributed to a combined action of the three antagonistic molecules. Electron microphotographs revealed the damage caused by L. lactis CRL 1584 supernatants to C. freundii cells. The addition of pure lactic acid, H(2)O(2) and bacteriocin to the culture media showed that each metabolite caused different morphological modifications in C. freundii, in agreement with the effect on viable cell counts. The results support the possibility that L. lactis CRL 1584 might be considered as a probiotic to be used in the prevention of RLS in raniculture.     

QuEChERS multiresidue method validation and mass spectrometric assessment for the novel anthranilic diamide insecticides chlorantraniliprole and cyantraniliprole

The gas-phase dissociation reactions of chlorantraniliprole (Rynaxypyr) and cyantraniliprole (Cyazypyr) have been studied in triple-quadrupole, ion trap, and orbitrap mass spectrometers equipped with electrospray and desorption electrospray ion sources, revealing the formation of odd-electron fragment ions, the structures of which were elucidated. The odd-electron fragments were unusually abundant, and their formation is proposed to occur via a tricyclic intermediate. The applicability of the QuEChERS multiresidue method for the quantitation of chlorantraniliprole and cyantraniliprole was also assessed in this study. Four matrices representative of oily, watery, acidic, and dry crop groups were tested, with a targeted limit of quantitation (LOQ) of 0.01 mg/kg. Average recoveries ranged between 87 and 107%, with relative standard deviations (RSD) of ≤ 8%. Linear calibration functions with correlation coefficients r > 0.99 were obtained. The study provides an expansion of the QuEChERS method to include anthranilic diamides and a mass spectrometric assessment for these two novel agrochemical active ingredients.     

Use of Anguilla anguilla for Biomonitoring Persistent Organic Pollutants (POPs) in Brackish and Riverine Waters in Central and Southern Italy

One of the aims of the Water Framework Directive 2000/60/CE is to assess the ecological status of water bodies in Europe in relation to priority contaminants, including some persistent organic pollutants (POPs). Recognizing the benefits of measuring hydrophobic compounds in biota tissues rather than in water, we used the European eel Anguilla anguilla in the present study as a bioindicator species for monitoring POPs in freshwater/brackish environments. We present the results of a contamination analysis of eels carried out in three Italian sites representative of different pollution levels: the last part of Tevere River, which flows through a very urbanized and industrialized area, Caprolace Lake and Lesina Lagoon, inside two different protected national parks. A very high pollution variability was recorded within each group of eels, in particular for those caught at Caprolace Lake. Due to this high variability, inter-population comparisons were carried out only between eels collected in Tevere River and Lesina Lagoon. PCBs values in Lesina Lagoon were two orders of magnitude lower than those measured in Tevere River, while no significant differences were found for levels of DDT compounds. Our results confirm the suitability of the eel as an indicator of persistent bioaccumulative pollutants in brackish environments and rivers where it can be easily caught, but we are sceptical of its use in deep lakes where its capture might be problematic considering standard operation procedures for fish sampling.     

Post-mortem recovery,in vitro maturation and fertilization of fallow deer (Dama dama,Linnaeus 1758) oocytes collected during reproductive and no reproductive season

Habitat degradation leads to small and fragmented populations, lower genetic variability and fertility overtime. Assisted reproductive techniques represent important tools to cope with the dramatic loss of biodiversity. Fallow deer (Dama dama), beyond its high commercial value and wide distribution, may represent the most suitable model to study endangered cervids. In this study, oocytes were recovered post-mortem from fallow deer during the breeding and no breeding seasons and were in vitro matured (IVM). The ability of cryopreserved thawed sperm samples recovered by electroejaculation from four adult males was tested by in vitro fertilization of IVM oocytes. The number of oocytes collected per ovary did significantly vary across seasons from 6.2 ± 0.92 during breeding season to 10.4 ± 1.26 during no breeding season (p = .006). Oocytes collected during the breeding season showed higher in vitro fertilization rate compared to the no breeding season (p = .045). However, no embryos reached the blastocyst stage. Semen samples obtained by electroejaculation were successfully cryopreserved, although the cryopreservation process negatively affected most kinetic parameters, mainly at 2 hr post-thawing. Moreover, the percentage of rapid spermatozoa significantly decreased between fresh samples and at 2 hr post-thawing, whereas the percentage of slow spermatozoa increased across the same period (p < .05). Our study provides the logistic steps for the application of assisted reproductive techniques in fallow deer and might be of great interest for genetic resource bank planning.     

Amount and speciation of N leached from a sandy soil fertilized with urea,liquid digestate,struvite and NH4-enriched chabazite zeolite-tuff

The large use and the bad management of fertilizers that are applied to soil for improving crop production have dramatically impaired soil, water, and air quality. To meet the requirements to reduce nitrogen (N) losses and all the related negative impacts on the environment and food production, it is mandatory to substitute or at least partially substitute the use of inefficient and unsustainable fertilizers with more efficient alternatives. The aim of this paper was to address the amount and speciation of the N released by a sandy soil fertilized with “slow-release fertilizers” and traditional fertilizers (urea and liquid digestate) by means of a series of column leaching experiments. The slow-release alternatives were represented by NH₄-enriched zeolitic tuff and struvite, both obtained by recovering the N from liquid digestate. The treatments consisted of sandy soil fertilized with (i) urea (U) (ii) liquid digestate (LD), (iii) NH₄-enriched zeolitic tuff (N-CHA) and (iv) struvite (STRV). Eight different flushing events were performed over 38 days, leachates were collected and analysed for total Kjeldahl N, organic-N, NH₄⁺-N, NO₃⁻-N, NO₂⁻-N and pH. U and LD lost the majority of N within the first 2 flushing events as organic N and NH₄⁺-N, respectively. On the other hand, STRV and N-CHA lost less N over the whole course of the experiment and with more balanced speciation. The mass balance outlined that after the experiment, native soil N was mined in U and LD treatments while in N-CHA and STRV a fraction of N from the fertilizers was still present. The results showed a slow release of N which can be used more efficiently in agricultural applications, minimizing the N losses.     

Genetic diversity in Borago officinalis germplasm as revealed by seed oils and AFLP polymorphism

Ten accessions of wild Borago officinalis were collected during missions in the course of a research activity carried out on plants producing nutraceutical compounds. The aim was to identify populations that produce a higher content of γ-linolenic acid, as this compound has important therapeutic properties and has been used in medical tests to treat rheumatoid arthritis, eczema, and psoriasis with good therapeutical results. The present study reports on the characterization of both the polyunsaturated fatty acid composition and on genetic diversity estimated by means of AFLP markers in several B. officinalis accessions collected in Southern Italy and grown in isolation by means of screen houses specifically designed for each single population. Polyunsaturated fatty acid were found in different amounts in all investigated accessions. The populations were very variable at the molecular level, and the analysis of relationships based on AFLP data, showed that similarity based on Jaccard’s index ranged between 0.330 and 0.742. Our investigations allowed us to identify a population that may be considered as a good source of GLA.     

Use of CLSA and SPME-headspace techniques followed by GC-MS analysis to extract and identify the floral odorants

Flowers of Ziziphus spina christi are known to be attractive for parasitoids and predators. In Y-tube olfactometer experiments, the dried flowers attracted significantly (p < 0.001) the female parasitoids Aphelinus abdominalis. The flower volatile compounds were analyzed to understand which compounds could be specifically responsible for this attractiveness. The volatile compounds of Ziziphus flowers were extracted by closed-loop-stripping-analysis (CLSA) and also by solid phase microextraction (SPME) followed by gas chromatography-mass spectrometry (GC-MS) analysis. The main chemical classes of the volatile compounds are aldehydes, monoterpene-alcohols, ketones and hydrocarbons. Flower extract and some specific compounds will be further tested for their responsiveness to predators and parasitoids in behavioural and electrophysiological experiments.     

Evaluation of the Lime-Cooking and Tortilla Making Properties of Quality Protein Maize Hybrids Grown in Mexico

Eleven experimental and three commercial white quality protein maize (QPM) hybrids and two regular endosperm controls were planted at Celaya, Guanajuato, Mexico with the aim of comparing grain physical characteristics, protein quality, lime-cooking and tortilla making properties. All genotypes were planted under irrigation using a density of 80,000 plants/ha and fertilized with 250 kg N-60 P-60 K per hectare. When compared with the controls these QPM genotypes had lower test (77.4 vs. 76.5 kg/hL) and 1,000 kernel weights (327 vs. 307 g), softer endosperm texture (2.5 vs. 1.8 where 1 = soft, 2 intermediate and 3 hard endosperm), lower protein (10.0 vs. 8.0%), higher nixtamal water uptake after 30 min lime-cooking (50.0 vs. 53.1% moisture) and lower pericarp removal scores. The lower thousand-kernel weight and softer endosperm texture observed in the QPM genotypes lowered the optimum lime-cooking time as estimated with regression equations. Most QPM genotypes had higher amounts of lysine, tryptophan and albumins/globulins when compared with the controls. QPMs HEC 424973, HEC 774986 and HEC 734286 had the best grain traits for nixtamalization and therefore the best potential for industrial utilization. The commercial use of these QPM hybrids should benefit Mexicans who depend on tortillas as the main staple.