Development of real-time PCR systems based on SYBR? Green I and TaqMan? technologies for specific quantitative detection of Phoma tracheiphila in infected Citrus

Real-time PCR assays based on SYBR? Green I and TaqMan? technologies were developed for in planta detection and quantification of Phoma tracheiphila, the mitosporic fungus causing ‘mal secco’ disease on citrus. Primers and a hybridization probe were designed on the basis of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The real-time PCR assays were compared with a classic isolation method in two separate experiments carried out on 6 and 24 month-old sour orange seedlings, artificially inoculated with a conidial suspension of the pathogen. Both technologies made it possible to follow the progression of infection by P. tracheiphila, enabling detection and quantification of the target fungus prior to the development of symptoms. The detection limit was 10 copies of the cloned target sequence and 15 pg of genomic DNA extracted from fungal spores. The values of the cycle threshold (Ct) were linearly correlated with the concentration of the target DNA, indicating that the method is suitable as a qualitative and quantitative assay. The presence of non-target fungal DNA had no effect on the specificity of the assay, but resulted in a 10-fold reduction of sensitivity. Total inhibition of the reaction occurred when conidia of the target pathogen were mixed with an organic soil substrate before extracting DNA using the standard protocol, while an alternative purification kit resulted in a significant decrease in sensitivity. Compared to classic methods, real-time PCR proved faster and easier to perform and showed a higher sensitivity. These results suggest that real-time PCR, based on both chemistries, has a great potential for early diagnosis of ‘mal secco’ disease and for quantitative estimation of fungal growth within host tissue.     

How to enhance crop production and nitrogen fluxes? A result-oriented scheme to evaluate best agri-environmental measures in Veneto Region,Italy

The cost-effectiveness of adopting agri-environmental measures (AEMs) in Europe, which combine agricultural productions with reduced N losses, is debated due to poorly targeted site-specific funding that is allocated regardless of local variability. An integrated DAYCENT model-GIS platform was developed combining pedo-climatic and agricultural systems information. The aim was to evaluate best strategies to improve N fluxes of agro-ecosystems within a perspective of sustainable intensification. Indicators of agronomic efficiency and environmental quality were considered. The results showed that agronomic benefits were observed with a continuous soil cover (conservation agriculture and cover crops), which enhanced nitrogen use efficiency (+17%) and crop yields (+34%), although in some cases these might be overestimated due to modelling limitations. An overall environmental improvement was found with continuous soil cover and long-term change from mineral to organic inputs (N_Leach < 10 kg ha^?1 a^?1, N-N₂O emissions < 1 kg ha^?1 a^?1, soil C stock > 45 Mg ha^?1), which were effective in the sandy soils of western and eastern Veneto with low SOM, improving the soil-water balance and nutrients availability over time. Results suggest that AEM subsidies should be allocated at a site-specific level that includes pedo-climatic variability, following a result-oriented approach.     

Activity of foliar extracts of cultivated eggplants against sclerotinia lettuce drop disease and their phytochemical profiles

Antifungal activity of plant-derived compounds can be exploited in disease management systems to improve sustainability and replace synthetic molecules. In this study, four crude hydroethanolic extracts of leaves, collected from Italian Solanum spp. landraces, were selected to evaluate their ability to suppress Sclerotinia minor Jagger, a great threat to lettuce production worldwide. In vitro fungal development was inhibited by Solanum melongena L. and S. aethiopicum L. extracts showing a dose-dependent correlation. At the highest concentration tested in the current experiments (45 mg mL^?1) antifungal activity caused up to 90% growth reduction. The exposure of the fungus to S. aethiopicum extracts incited pronounced changes in the hyphal morphology as observed under light microscopy. Consistently, under laboratory conditions, in planta application of the active extracts on lettuce significantly reduced Sclerotinia drop disease in comparison to non-treated controls. Phytochemical composition was determined by liquid chromatography/mass spectrometry (LC/MS) analyses. Four secondary metabolites differentially present in the extracts, identified as n-caffeoylputrescine, chlorogenic acid, isoquercitrin and solasodoside A, are hypothesized to play a crucial role in mechanisms underlying biological effects of extracts. PCA analysis showed positive correlations of these compounds with the overall control ability of the extracts. The results indicated that foliar material from cultivated eggplant could be suitable to produce biological-based remedies for controlling plant diseases.     

Meat quality and intramuscular fatty acid composition of Catria Horse

In order to extend scientific knowledge on autochthonous Italian equine meat, the physical–chemical parameters of Catria Horse Longissimus thoracis (LT) muscle and its nutritional characteristics have been investigated. Ten steaks of Catria foal raised at pasture and fattened indoors for 2 months were dissected, and LT muscle was analyzed for chemical composition, total iron, drip loss, colorimetric characteristics, intramuscular fat, fatty acid profile and nutritional indexes. Steak dissection showed that LT muscle accounted for 36.78% and fat accounted for 9.19% of weight of steak. Regarding chemical composition, protein and fat content was 20.31% and 2.83%, respectively. Total iron content (1.95 mg/100 g) was lower than data reported in the literature. Color parameters showed a luminous and intense red hue muscle. The sum of unsaturated fatty acid composition (50.3%) was higher than the sum of saturated fatty acids (46.64 %). The fatty acid profile and nutritional values of Catria Horse meat could be modified adopting extensive rearing systems and grazing. The data suggests that further investigation on the composition of Catria Horse meat should be carried out to valorize this autochthonous breed, reared in sustainable livestock systems, and its meat in local short‐chain systems.     

Selection,characterization and genetic analysis of laboratory mutants of <Emphasis Type="Italic">Botryotinia fuckeliana</Emphasis> (<Emphasis Type="Italic">Botrytis cinerea</Emphasis>) resistant to the fungicide boscalid

Resistance to the fungicide boscalid in laboratory mutants of Botryotinia fuckeliana (Botrytis cinerea) was investigated. The baseline sensitivity to boscalid was evaluated in terms of colony growth (EC₅₀ = 0.3–3 μg ml⁻¹; MIC = 10–30 μg ml⁻¹) and conidial germination (EC₅₀ = 0.03–0.1 μg ml⁻¹; MIC = 1–3 μg ml⁻¹) tests. Mutants were selected in vitro from wild-type strains of the fungus on a fungicide-amended medium containing acetate as a carbon source. Mutants showed two different levels of resistance to boscalid, distinguishable through the conidial germination tests: low (EC₅₀ ∼ 0.3 μg ml⁻¹, ranging from 0.03 to 1 μg ml⁻¹; MIC > 100 μg ml⁻¹) and high (EC₅₀ > 100 μg ml⁻¹) resistance. Analysis of meiotic progeny from crosses between resistant mutants and sensitive reference strains showed that resistant phenotypes were due to mutations in single major gene(s) inherited in a Mendelian fashion, and linked with both the Daf1 and Mbc1 genes, responsible for resistance to dicarboximide and benzimidazole fungicides, respectively. Gene sequence analysis of the four sub-units of the boscalid-target protein, the succinate dehydrogenase enzyme, revealed that single or double point mutations in the highly conserved regions of the iron-sulphur protein (Ip) gene were associated with resistance. Mutations resulted in proline to leucine or phenylalanine replacements at position 225 (P225L or P225F) in high resistant mutants, and in a histidine to tyrosine replacement at position 272 (H272Y) in low resistant mutants. Sequences of the flavoprotein and the two transmembrane sub-units of succinate dehydrogenase were never affected.     

Isolation and characterization of Avenin-like protein type-B from durum wheat

Proteomic techniques were applied for the investigation of “avenin-like protein type-B” in tetraploid wheat. Protein was extracted from endosperm, according to a classical chromatographic procedure and its identity confirmed by liquid chromatography-tandem mass spectrometry analysis. The characterization of avenin-like protein type-B was in good agreement with the gene-derived sequence, with the exception of glutamine as N-terminus. The presence in the glutenin extract and the high number of cysteine residues suggested that avenin-like protein type-B is integrated into gluten polymers via inter-chain disulphide bonds. This raises the question of whether the protein could play a role in determining the functional properties of gluten.     

Effects of two fractions of inspired oxygen on lung aeration and gas exchange in cats under inhalant anaesthesia

ObjectiveTo compare the effects of two fractions of inspired oxygen (FiO₂) (0.4 and 1) on lung aeration and gas exchange during general anaesthesia in cats.Study designRandomized, blinded, controlled study.AnimalsThirty healthy, mixed breed, client owned female cats.Materials and methodsCats were premedicated intramuscularly with acepromazine (0.03 mg kg^?1) and medetomidine (0.015 mg kg^?1). Anaesthesia was induced with propofol (5 mg kg^?1) and, after orotracheal intubation, maintained with isoflurane carried by either 100% oxygen (G100, n = 15) or an oxygen-air mixture with 40% oxygen (G40, n = 15). All cats were placed in dorsal recumbency and breathed spontaneously throughout the entire procedure. Following surgery (ovariectomy), a spiral computed tomography (CT) of the thorax was performed, arterial oxygen (PaO₂) and carbon dioxide (PaCO₂) tensions were measured and alveolar-arterial gradient of oxygen [P(A-a)O₂] calculated. The CT images were analysed for lung aeration by the analysis of radiograph attenuations (Hounsfield units, HU), according to the following classification: hyperinflated area (-1000 to -900 HU), normally aerated area (-900 to -500 HU), poorly aerated area (-500 to -100 HU) and non-aerated area (-100 to +100 HU). The groups were compared using one-way anova.ResultsCompared to G100, the normally-aerated lung area was significantly greater and the poorly-aerated and non-aerated areas were significantly smaller in G40. PaCO₂ was similar in both groups. PaO₂ and P(A-a)O₂ were significantly higher in G100. In both groups, pulmonary atelectasis developed preferentially in the caudal lung fields.ConclusionIn cats anaesthetised with isoflurane, the administration of an FiO₂ of >0.9 significantly impaired lung aeration and gas exchange as compared to an FiO₂ of 0.4.Clinical relevanceAn FiO₂ of 0.4 may better preserve lung aeration and gas exchange in anaesthetised spontaneously breathing cats but monitoring is essential to ensure oxygenation is adequate.