The Comet assay for detection of DNA damage in canine sperm

Sperm DNA integrity is a fundamental prerequisite in fertilization and embryo development. Among DNA integrity tests, the Comet assay is an accurate and sensitive test for the detection of sperm oxidative damage. The aim of this work was to evaluate sperm oxidative damage using the Comet assay and to study the correlation between Comet and routine assays for the evaluation of semen quality. Dogs were divided in two groups: group A (n = 6), comprising dogs with abnormal spermiogram, that is astheno‐, terato‐ or oligoasthenoteratozoospermic (OAT); and group B (n = 8), comprising normospermic dogs. The distribution of sperm oxidative damage was significantly different between the two groups (p = .001): group A—median: 31.55%, interquartile range (IQR): 30.18–38.01; group B—median: 0.90%, IQR: 0.65–1.96. The correlation between oxidative damage and abnormal morphology was high (r = .846; p < .001). There was a negative correlation between progressive motility and oxidative damage (r = ?.792; p = .001). Basal and oxidative DNA damage of spermatozoa are increased in dogs with non‐normospermic semen. In conclusion, and considering the elevated correlation with classical tests of sperm quality, the Comet assay has ample potential for clinical and research purposes in dogs.     

Antibodies to Borrelia burgdorferi in New England horses: serologic survey

Twelve of 50 randomly selected horses from areas endemic for Borrelia burgdorferi had indirect fluorescent antibody titers of 1:8 to 1:2,048 against B burgdorferi. One of 50 horses from nonendemic areas had a titer of 1:8. This difference in the number of horses seropositive for B burgdorferi (P less than 0.002) and our finding that seropositive horses did not have agglutinating antibodies against potentially cross-reacting Leptospira spp indicated that horses in endemic areas were exposed to B burgdorferi and that the spirochete induced an antibody response in the horses.     

Biometrical, biological, biochemical and molecular characteristics of Meloidogyne incognita isolates and related species

Meloidogyne incognita is one of the most polyphagous species of root-knot nematodes occurring in Brazil and worldwide. Eight M. incognita isolates were studied, representing two enzymatic phenotypes (esterase and malate desydrogenase: I1/N1, I2/N1) and four cryptic Meloidogyne sp.1 (S2/N1) isolates, representing one cytological type (3n?=?40–46). Three M. hispanica isolates (Hi3/N1, 2n?=?32–36) and two of an atypical Meloidogyne sp.2 (S2a/N3, 3n?=?40–44) were included in this study for comparison. All isolates were tested with three M. incognita-specific molecular markers. The primer pairs B06F/R, miF/R and incK14F/R amplified three species-specific fragments of 1,200?bp, 955?bp and 399?bp, respectively for M. incognita and Meloidogyne sp.1 isolates. No amplification occurred in the M. hispanica and Meloidogyne sp.2 isolates, except with primers miF/R (1,650?bp). The genetic variability of the Meloidogyne spp. isolates was evaluated, using RAPD and ISSR markers. The phylogenetic analyses revealed two strongly supported monophyletic clades: clade I, consisting of M. hispanica and the atypical Meloidogyne sp.2 isolates, and clade II, clustering together all M. incognita and the Meloidogyne sp.1 isolates. Considering the biometrical, cytological and molecular approaches, it was possible to conclude that the isolates with three enzymatic phenotypes (I1/N1, I2/N1 and S2/N1) presented the characteristics described for M. incognita. Some correlations were detected between the isozymatic phenotypes and the tree topology (S2a/N3, Hi3/N1, I1/N1, S2/N1), but no strict correlation could be observed for the phenotype I2/N1 and one isolate of S2/N1. Morphologically, the Msp.2 isolates differ from M. incognita and M. hispanica by the female stylet features presenting straight cone tip and round pear shaped knobs, posteriorly sloping. The results of this study suggested that the Msp.2 isolates with phenotypes S2aN3 belong to a new or an unidentified species closely related to M. hispanica.     

Genetic variability evaluation in a Moroccan collection of barley, Hordeum vulgare L., by means of storage proteins and RAPDs

The genetic variation existing in a set of barley (Hordeum vulgare L.) landrace samples recently collected in Morocco was estimated. Two kinds of genetic markers, seed storage proteins (hordeins) and random amplified polymorphic DNA (RAPD), were used. Only six out of 31 landraces were subjected to RAPD analysis. Both kinds of markers, RAPD and storage proteins, yielded similar results, showing that the level of variation observed in Moroccan barley was high: all landraces showed variability; 808 different storage protein patterns (multilocus associations) were observed among 1897 individuals (2.32 seeds per association, on average) with an average of 43 multilocus associations per accession. In general, genetic variation within accessions was higher than between accessions. The 100 polymorphic RAPD bands generated by 21 effective primers were able to generate enough patterns to differentiate between uniform cultivars and even between individuals in variable accessions. One of the aims of this work was to compare the effectiveness of RAPD versus storage protein techniques in assessing the variability of genetic resource collections. On average hordeins were more polymorphic than RAPDs: they showed more alternatives per band on gels and a higher percentage of polymorphic bands, although RAPDs supply a higher number of bands. Although RAPD is an easy and standard technique, storage protein analysis is technically easier, cheaper and needs less sophisticated equipment. Thus, when resources are a limiting factor and considering the cost of consumables and work time, seed storage proteins must be the technique of choice for a first estimation of genetic variation in plant genetic resource collections.     

Sodicity and salinity in a Brazilian Oxisol cultivated with sugarcane irrigated with wastewater

Changes in soil sodicity-salinity parameters are one of the most characteristic alterations after treated sewage effluent (TSE) irrigation in agro-systems. Considering the importance of these parameters for agricultural management, as well as the economical value of sugarcane for Brazil, the present study aimed at evaluating effects on soil sodicity and salinity under tropical conditions over 16 months of TSE irrigation in a sugarcane plantation at Lins, São Paulo State, Brazil. Soil samplings were carried out in February 2005 (before planting), December 2005 (after 8 months of TSE irrigation) and September 2006 (after 16 months of TSE irrigation) following a complete block design with four treatments and four replicates. Treatments consisted of: (i) control, without TSE irrigation; (ii) T100, T150 and T200, with TSE irrigation supplying 100% (0% surplus, total of 2524 mm), 150% (50% surplus, total of 3832 mm) and 200% (100% surplus, total of 5092 mm) of crop water demand, respectively. Compared to initial soil conditions, at the end of the experiment increases of exchangeable sodium (from 2.4 to 5.9 mmol_c kg⁻¹), exchangeable sodium percentage (ESP) (from 8 to 18%), soluble Na (from 1.4 to 4.7 mmol L⁻¹) and sodium adsorption ratio (SAR) of soil solution (from 3.6 to 12.6 (mmol L⁻¹)^0.5) were found in the soil profile (0-100 cm) as an average for the irrigated plots due to high SAR of TSE. Associated with the increments were mostly significant increases in clay dispersion rates at depths 0-10, 10-20 and 20-40 cm. Electrical conductivity (EC) of soil solution increased during the TSE irrigation period whereas at the end of the experiment, after short term discontinuation of irrigation and harvest, EC in the topsoil (0-10 and 10-20 cm) decreased compared to the previous samplings. Moreover, despite increasing sodicity over time mainly insignificant differences within the different irrigated treatments were found in December 2005 and September 2006. This suggests that independent of varying irrigation amounts the increasing soil sodicity over time were rather caused by the continuous use of TSE than by its quantity applied. Moreover, also plant productivity showed no significant differences within the TSE irrigated plots. The study indicates that monitoring as well as remediation of soil after TSE irrigation is required for a sustainable TSE use in order to maintain agricultural quality parameters.     

Genomic characterization of multidrug-resistant extraintestinal pathogenic Escherichia coli isolated from grain culture soils

Multidrug-resistant (MDR) Escherichia coli, mainly extraintestinal pathogenic E. coli (ExPEC), has been widely reported in infections worldwide. In agricultural soils, manure is a hotspot for the dissemination of antimicrobial resistance genes (ARGs) and pathogenic bacteria; however, MDR bacteria have also been reported in soils with no history of manure use. In addition, cross-resistance and co-resistance have been described as responsible for the metal-driven selection of bacteria resistant to antimicrobials. Therefore, the aim of this study was to analyze three MDR E. coli isolates obtained from Brazilian grain culture soil samples with no history of manure use by whole-genome sequencing. The MDR E. coli isolates were recovered from soils from corn and coffee fields, and presented resistance to β-lactams, quinolones, aminoglycosides, tetracyclines, sulphonamides, and dihydrofolate reductase inhibitor. Resistome analysis showed ARGs to several antimicrobials (i.e., β-lactams, tetracyclines, aminoglycosides, sulphonamides, trimethoprim, phenicols, fosfomycin, and macrolides) as well as several metal resistance genes and antibacterial biocide resistance genes. In addition, known mutations in quinolone-resistance-determining regions of GyrA (Ser83Leu and Asp87Asn), ParE (Ser458Thr), and ParC (Ser80Ile) were also detected. Virulome analysis showed the presence of virulence genes (lpfA, mcmA, gad, mchF, iroN, cma, and iss) associated with ExPEC. Multidrug-resistant ExPEC isolates were assigned to phylogenetic group B1. The presence of MDR B1-ExPEC in soil samples shows the ability of these isolates to survive in soils. This study reports for the first time some sequence types (i.e., ST345, ST448, and ST1146) of MDR E. coli in Brazilian soils. Therefore, these findings contribute to the monitoring of antimicrobial resistance and surveillance studies based on whole-genome sequencing worldwide.     

Soil carbon sequestration with continuous no-till management of grain cropping systems in the Virginia coastal plain

Carbon sequestration in agroecosystems represents a significant opportunity to offset a portion of anthropogenic CO₂ emissions. Climatic conditions in the Virginia coastal plain and modern production practices make it possible for high annual photosynthetic CO₂ fixation. There is potential to sequester a substantial amount of C, and concomitantly improve soil quality, with the elimination of tillage for crop production in this region. The objectives of our research were to: (1) measure C sequestration rate with continuous no-till management of grain cropping systems of the Virginia middle coastal plain; (2) determine the influence of biosolids application history on C content and its interaction with tillage management; and (3) evaluate the impact of continuous no-till C stratification as an indicator of soil quality. Samples were collected from 63 sites in production fields using a rotation of corn (Zea mays L.)–wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.)/soybean double-crop (Glysine max L.) across three soil series [Bojac (coarse-loamy, mixed, semiactive, thermic Typic Hapludults), Altavista (fine-loamy, mixed semiactive, thermic Aquic Hapludults), and Kempsville (fine-loamy, siliceous, subactive, thermic Typic Hapludults)] with a history of continuous no-till management ranging from 0 to 14 years. Thirty-two of the sites had a history of biosolids application. Five soil cores were collected at each site from 0–2.5, 2.5–7.5 and 7.5–15 cm and analyzed for bulk density and soil C. Bulk density in the 0–2.5 cm layer decreased and C stratification ratio (0–2.5 cm:7.5–15 cm) increased with increasing duration of continuous no-till due to the accumulation of organic matter at the soil surface. A history of biosolids application resulted in an increase of 4.19 ± 1.93 Mg C ha⁻¹ (0–15 cm). Continuous no-till resulted in the sequestration of 0.308 ± 0.280 Mg C ha⁻¹ yr⁻¹ (0–15 cm). Our results provide quantitative validation of the C sequestration rate and improved soil quality with continuous no-till management in the region using on-farm observations.     

Outbreak of shoot blight and dieback of Eucalyptus spp., caused by Pseudoplagiostoma eucalypti in Brazil

An outbreak of a new and severe disease was observed in Eucalyptus plantations of Bahia state, Brazil. An Ascomycota fungus has been frequently associated with the main symptoms of the disease namely leaf spot, branch cankers, shoot blight, defoliation, and dieback. Based on morphological characteristics, phylogenetic analysis (ITS and TEF-1α genes), and pathogenicity test on Eucalyptus plants, Pseudoplagiostoma eucalypti was identified as the causal agent of the disease. Although P. eucalytpi has been known from in Brazil since 1998, this is the first report of it causing severe disease and die-back on Eucalyptus spp. and we also record new symptoms associated with the pathogen.