Interaction between root rot basidiomycetes and Phytophthora species on pedunculate oak

Forest declines are usually complex multifactorial phenomena that involve interactions between different factors. The possible interaction between different types of mycelial pathogens was investigated through artificial inoculation of oak seedlings, involving two root rot basidiomycetes, Collybia fusipes and Armillaria mellea, and two Phytophthora species, P. cinnamomi and P. cambivora. These pathogens were inoculated onto young Quercus robur saplings in greenhouse conditions, either alone or combining a root rot basidiomycete with a Phytophthora species. Three out of the four Phytophthora spp.*root rot basidiomycete combinations tested resulted in significantly greater damage to the oak host than the sum of the damages induced by the individual pathogens. This positive interaction could be significant in oak decline syndrome.     

Subolesin/akirin orthologs from Ornithodoros spp. soft ticks: cloning, RNAi gene silencing and protective effect of the recombinant proteins

Subolesin/akirin is a well characterized protective antigen highly conserved across vector species and thus potentially useful for the development of a broad-spectrum vaccine for the control of arthropod infestations including hard ticks, mosquitoes, sand flies and the poultry red mite Dermanyssus gallinae. Soft ticks could be also targeted by this vaccine if proved that the soft tick subolesin orthologs are conserved and induce protective immune responses too. However, to date no soft tick subolesin orthologs have been fully characterized nor tested as recombinant antigens in vaccination trials. The objectives of the present work were to clone and characterize the subolesin orthologs from two important vector species of soft ticks as Ornithodoros erraticus and O. moubata, to evaluate the effect of subolesin gene silencing by RNAi, and to test the protective value of the recombinant antigens in vaccination trials. The obtained results demonstrate that both soft tick subolesins are highly conserved showing more than 69% and 74% identity with those of hard ticks in their nucleotide and amino acid sequences, respectively. Additionally, we demonstrate that both soft ticks possess fully operative RNAi machinery, and that subolesin gene silencing by dsRNA injection inhibits oviposition indicating the involvement of subolesin in tick reproduction. Finally, vaccination with the recombinant soft tick subolesins induced a partial protective effect resulting in the reduction of the oviposition rate. These preliminary results encourage further studies on the use of recombinant subolesins as vaccines for the control of soft tick infestations, either alone or in combination with other specific molecules.     

Use of touch-down polymerase chain reaction to enhance the sensitivity of Mycobacterium bovis detection.

The confirmatory diagnosis of Mycobacterium bovis (M. bovis) in animal samples is carried out by culture in Stonebrink media. However, culture is very slow because of the extremely long duplication time of the bacillus and difficult because of the scarcity of bacilli in diagnostic samples. This study describes the development of a single-tube touch-down polymerase chain reaction (PCR) protocol for the detection of M. bovis using primers that target the IS6110 element. Spiked water and milk as well as routine diagnostic samples (milk and nasal swabs) from M. bovis-positive cattle were tested. This protocol allows the rapid and sensitive detection of M. bovis in bovine samples by enhancing the sensitivity of standard PCR amplification.     

Pharmacokinetics of azithromycin after i.v. and i.m. administration to sheep

The pharmacokinetics (PK) of azithromycin after i.v. and i.m. injection at a single dosage of 20 mg/kg bodyweight was studied in sheep. Blood samples were collected from the jugular vein until 120 h after dosing for both routes. Plasma concentrations of azithromycin were determined by bioassay. The plasma concentration-time data of azithromycin best fitted a three-compartment model after i.v. administration and a two-compartment model with first-order absorption after i.m. administration. The elimination half-life (t(1/2lambdaz)) was 47.70 +/- 7.49 h after i.v. administration and 61.29 +/- 13.86 h after i.m. administration. Clearance value after i.v. dosing was 0.52 +/- 0.08 L/kg.h. After i.m. administration a peak azithromycin concentration (C(max)) of 1.26 +/- 0.19 mg/L was achieved at 1.24 +/- 0.31 h (t(max)). Area under the curve (AUC) were 38.85 +/- 5.83 mg.h/L and 36.03 +/- 1.52 mg.h/L after i.v. and i.m. administration respectively. Bioavailability obtained after i.m. administration was 94.08 +/- 11.56%. The high tolerability of this i.m. preparation and the favourable PK behaviour such as the long half-life and high bioavailability make azithromycin likely to be effective in sheep.     

Triploid Induction in the Yellowtail Tetra,Astyanax altiparanae,Using Temperature Shock: Tools for Conservation and Aquaculture

Triploidization is an interesting tool to produce sterile fish. In the yellowtail tetra, Astyanax altiparanae, this can be applied for aquaculture and surrogate technologies. In this study, we compared the efficacy of cold (2 C) or heat shock (38 C, 40 C, and 42 C) on triploid induction in the yellowtail tetra. The eggs were treated with cold or heat shock, 2 min postfertilization (30 min in cold shock or 2 min in heat shock). Intact embryos served as the control group. Ploidy status was confirmed by karyotyping, flow cytometry, and nuclear diameter of erythrocytes. The hatching rate decreased after cold shock (12.69 ± 15.76%) and heat shock at 42 C (0.35 ± 0.69%) in comparison with the control group (63.19 ± 16.82%). At 38 C and 40 C, hatching rates (61.29 ± 17.73% and 61.75 ± 22.1%, respectively) were not decreased. Only one triploid arose at 38 C (1/80). At 40 C, a high number of triploids arose (72/78). At 42 C, very few embryos developed into the hatching stage. A large number of haploid individuals arose after cold shock (61/75), with only one triploid. Our results indicate that heat shocking of embryos at 40 C is optimum for triploid production in the yellowtail tetra.     

Evaluation of the API 50CH and API ZYM systems for rapid characterization of Clavibacter michiganensis subsp. sepedonicus, causal agent of potato ring rot

API 50CH and API ZYM systems were used to characterize fifty-three strains of Clavibacter michiganensis subsp. sepedonicus from different geographic locations and several reference strains of the same and different species, including other potato pathogens. Clavibacter michiganensis subsp. sepedonicus strains displayed a high level of homogeneity, both in carbohydrate utilization and in enzymatic activity. Using API 50CH and API ZYM it was possible to differentiate C. michiganensis subsp. sepedonicus strains from the remaining taxa analysed in this study, which included representative strains of the other subspecies of C. michiganensis as well as other bacterial pathogens affecting potatoes. Therefore, these systems could be used as an effective method to characterize C. michiganensis subsp. sepedonicus. Such a procedure would constitute an alternative system to the conventional nutritional and physiological identification tests currently included in the official methods employed in the European Union to detect and identify this bacterium. The results obtained with the API systems agreed with the current taxonomic classification of C. michiganensis, clearly separating sepedonicus from the other subspecies belonging to this species.     

Organic amendments affecting sorption, leaching and dissipation of fungicides in soils

Metalaxyl and tricyclazole are two fungicides widely used in Spain in vineyard and rice crops respectively. In this study an investigation has been made of the effect of three organic amendments [two commercial amendments, solid fertiormont (SF) and liquid fertiormont (LF), and a residue from the olive oil production industry, alperujo (OW)] on fungicide fate in soils. Changes in soil porosity on amendment were studied by mercury intrusion porosimetry, sorption-desorption studies were performed by the batch equilibration method, dissipation of metalaxyl and tricyclazole in the soil was studied at - 33 kPa moisture content and 20 degrees C and leaching was studied in hand-packed soil columns. Amendments with SF and LF reduced soil porosity, while OW increased porosity through an increase in pore volume in the highest range studied. Tricyclazole sorbed to soils to a much higher extent than metalaxyl. With some exceptions, sorption of both fungicides increased on amendment, especially in the case of SF-amended soils, which rendered the highest K(oc) values. In soils amended with the liquid amendment LF, sorption either remained unaffected or decreased, and this decrease was much higher in the case of metalaxyl and a soil with 70% clay. In this clay soil, amendment with OW, of very high soluble organic matter content, also decreased metalaxyl sorption. Tricyclazole is more persistent in soil than metalaxyl, and both fungicides were found to be more persistent in amended soils than in unamended soils. Leaching of metalaxyl and tricyclazole in soil columns was inversely related to sorption capacity. The low recoveries of tricyclazole in leachates and in soil columns when compared with metalaxyl, a less persistent fungicide, were attributed to diffusion into micropores and to increase in sorption with residence time in the soil, both processes favoured by the low mobility of tricyclazole.