Tissue distribution of Red Spotted Grouper Nervous Necrosis Virus (RGNNV) genome in experimentally infected juvenile European seabass (Dicentrarchus labrax)

The distribution of viral genome in the tissues of juvenile European seabass (Dicentrarchus labrax) during the course of a Red Spotted Grouper Nervous Necrosis Virus (RGNNV) infection has not yet been described. The present study addresses this and indicates which target organs may be involved in viral replication. This information should enable more accurate detection of virus in asymptomatic carriers, and in turn help to control the spread of the disease. The aim of this study was to examine the pattern of expression of viral genomic segments RNA1 and RNA2, using two absolute real-time PCRs (RT-qPCR), over the course of a RGNNV infection after administering the virus by intramuscular injection. In situ hybridization was also used to locate the RNA2 viral segment in different organs throughout the infection. The experimental challenge provoked an acute form of viral nervous necrosis (VNN), with a resulting cumulative mortality of 37%. The RT-qPCRs designed allowed the detection of both genomic segments in all the organs tested (nervous and non-nervous tissues) at all sampling times examined. The highest viral RNA copy number was found in eyes, although viral replication appeared to begin in the brain. Viral replication was also recorded in pooled internal organs and in caudal fin. However, the increase in the viral RNA copy number in these organs did not result in an increased viral titre, which may indicate that a productive infection does not take place in non-nervous tissues, possibly due to a failure in a viral post-replication step.     

Evidence of Migration and Endophytic Presence of Agrobacterium tumefaciens in Rose Plants

Agrobacterium tumefaciens was isolated from stem tumors of several rose cultivars showing that the bacterium is the causal agent of aerial galls in rose plants. No differences were observed in the characteristics of the Agrobacterium isolates from crown or aerial galls. Stem inoculation of ten rose cultivars showed that all of them were susceptible to A. tumefaciens but differences in the size of the resulting tumors were observed. The movement of A. tumefaciens in rose plants was demonstrated using two wild type strains and two antibiotic resistant mutants. Three months after inoculation, the inoculated strains were recovered in the roots, crown and below and above the inoculation site but low numbers of pathogenic Agrobacterium cells were isolated. New tumors appeared in 5% of the noninoculated wounds. A. tumefaciens was isolated from the stem at different distances from the tumor in naturally infected plants. In symptomless commercial plants, the isolation from the roots, crown and at different stem levels demonstrated the existence of systemic and latent infections in rose. Direct isolation using a nonselective and selective media with or without a previous enrichment step were efficient methods for isolating tumorigenic Agrobacterium from the different parts of rose plants.     

A cadaver study comparing two approaches for performing maxillary nerve block in dogs

Objective To compare the success by inexperienced anaesthetists of using a modified infraorbital approach to the maxillary nerve with the traditional percutaneous approach. Study design Prospective, randomized, blinded controlled study. Animals Heads from 37 euthanized Beagle and Beagle cross dogs. Methods Four anaesthetists were recruited to perform two different approaches to block the maxillary nerve of the cadavers. The infraorbital (I) approach advanced an intravenous catheter along the infraorbital canal. Earlier measurements from scans of similar heads were used to assess suitable catheter size. The percutaneous (P) approach introduced a needle percutaneously just below the ventral border of the zygomatic arch. The side of the head where the technique was to be performed was randomized. A total volume of 0.5 mL methylene blue was injected in each approach. After completion of injections, head dissections were performed by an investigator unaware of the approach used and staining of the maxillary and pterygopalatine nerves was evaluated. Chi squared analysis examined the relationship between the methods (p < 0.05). Complications related to the techniques, such as intravascular/intraneural injection and location of the dye, were evaluated macroscopically. Results Maxillary nerve staining >6 mm was found in 64.9% (I) versus 21.6% (P) attempts; staining <6 mm was found in 27% (I) versus 21.6% (P); and no nerve staining 8.1% (I) versus 56.8% (M). Pterygopalatine nerve staining was found in 70% (I) versus 21% (P). The infraorbital approach demonstrated significantly higher maxillary and pterygopalatine nerve staining compared to the percutaneous approach (p = 0.001 for both nerves). No evidence of intravascular/intraneural injections was found. Conclusion and clinical relevance The infraorbital approach was more successful than the percutaneous approach when performed by inexperienced anaesthetists. No macroscopic complications were observed.     

Protein Composition of Seminal Plasma in Fractionated Stallion Ejaculates

Seminal plasma (SP) contains several types of compounds derived from the epididymides and accessory glands. The aim of this study was to examine the protein composition of different ejaculate fractions. Trial I: fractionated ejaculates were collected from two normal and two subfertile stallions. Samples containing pre‐sperm fluid and the first sperm‐rich jets (HIGH‐1), the main sperm‐rich portion (HIGH‐2), the jets with low sperm concentrations (LOW), and a combined whole‐ejaculate (WE) sample was centrifuged, and the SP was filtered and frozen. A part of each SP sample was stored (5°C, 24 h) with spermatozoa from HIGH‐2 and skim milk extender. Sperm motility was evaluated after storage in extender mixed with the stallion’s own SP or SP from one of the other stallions (sperm from a normal stallion stored in SP from a subfertile stallion and vice versa). Protein composition was analysed using reverse‐phase liquid chromatography (RP‐HPLC), N‐terminal sequencing and mass spectrometry. The area‐under‐the‐curve (AUC) was used for quantitative comparison of proteins within fractions. Trial II: semen samples were collected from seven stallions. Fractions with the highest (HIGH) and lowest (LOW) sperm concentrations and WE samples were examined using SDS‐PAGE and densitometry. No significant differences emerged between fractions in the AUC‐values of the Horse Seminal Protein‐1 (HSP‐1) and HSP‐2 peaks, or the peak containing HSP‐3 and HSP‐4 (HSP‐3/4). Levels of HSP‐1, HSP‐2 and HSP‐3/4 were not significantly correlated with total sperm motility, progressive sperm motility or average path velocity after storage. Significant differences between ejaculate fractions in the amount of different protein groups present in SP were not found in Trial I; but in Trial II, the proteins in the 60–70 kDa range were more abundant in LOW than in HIGH and WE, indicating that this band contained proteins derived mainly from the seminal vesicles, which produce most of the SP in LOW.     

Establishment of Retama sphaerocarpa L. seedlings on a degraded semiarid soil as influenced by mycorrhizal inoculation and sewage‐sludge amendment

A field experiment was carried out to evaluate the effectiveness of mycorrhizal inoculation with three arbuscular mycorrhizal (AM) fungi (Glomus intraradices Schenck & Smith, Glomus deserticola (Trappe, Bloss. & Menge), and Glomus mosseae (Nicol & Gerd.) Gerd. & Trappe) and the addition of composted sewage sludge (SS) with respect to the establishment of Retama sphaerocarpa L. seedlings, in a semiarid Mediterranean area. Associated changes in soil chemical (nutrient content and labile carbon fractions), biochemical (enzyme activities), and physical (aggregate stability) parameters were observed. Six months after planting, both the addition of composted SS and the mycorrhizal‐inoculation treatments had increased total N content, available‐P content, and aggregate stability of the soil. Values of water‐soluble C and water‐soluble carbohydrates were increased only in the mycorrhizal‐inoculation treatments. Rhizosphere soil from the mycorrhizal‐inoculation treatments had significantly higher enzyme activities (dehydrogenase, protease‐BAA, acid phosphatase, and β‐glucosidase) than the control soil. In the short‐term, mycorrhizal inoculation with AM fungi was the most effective treatment for enhancement of shoot biomass, particularly with G. mosseae (about 146% higher with respect to control plants). The addition of the composted SS alone was sufficient to restore soil structural stability but was not effective with respect to improving the performance of R. sphaerocarpa plants.     

Evaluation of preflooding effects on iron extractability and phytoavailability in highly calcareous soil in containers

Previous pot cropping and laboratory incubation experiments were consistent with field observations showing that temporary flooding before cropping can increase the availability of soil Fe to plants. To study the effect of temporary flooding on changes in soil Fe phytoavailability we used 24 highly calcareous, Fe chlorosis–inducing soils to carry out a pot experiment where peanut and chickpea were successively grown after flooding for 30 d. At the end of the cropping experiment, the preflooded soil samples exhibited higher concentrations of acid oxalate‐, citrate/ascorbate‐ and diethylenetriaminepentacetic acid (DTPA)–extractable Fe (Fe_ox, Fe_ca, and Fe_DTPA, respectively) than the control (nonflooded) samples. Also, Fe_ox and Fe_ca exhibited no change by effect of reflooding of the cropped soils or three wetting–drying cycles in freeze‐dried slurries of soils previously incubated anaerobically for several weeks. Leaf chlorophyll concentration (LCC) in both peanut and chickpea was greatly increased by preflooding. The best predictor for LCC was Fe_ox, followed by Fe_ca and Fe_DTPA. The LCC–soil Fe relationships found suggest that the Fe species extracted by oxalate and citrate/ascorbate from preflooded soils were more phytoavailable than those extracted from control soils. However, the increased phytoavailability of extractable Fe forms was seemingly limited to the first crop (peanut). Flooding dramatically increased Fe_DTPA; however, high Fe_DTPA levels did not result in high LCC values, particularly in the second crop. Therefore, this test is a poor predictor of the severity of Fe chlorosis in preflooded soils.     

Spatial variability of short-term effect of tillage on soil penetration resistance

The effect of tillage on soil properties varies within field due to spatial variability of soils. Mapping changes of soil penetration resistance (PR) would be useful to understand and assess tillage practices to alleviate soil compaction. The objectives were to model the short-term effect of tillage on PR and its spatial structure, and to delineate homogeneous zones based on soil response in a Typic Argiudoll previously managed under no-till. A grid sampling for PR and soil water content (SWC) were performed before and after chiselling. Spatial analysis was performed on the effect of tillage on PR data by 10 cm layers and homogeneous zones were delineated by k-means cluster analysis. The effect of tillage was ?0.33 MPa in 10–20 and 20–30 cm layer. No differences of PR were found at 0–10 cm. Short range (5–7 m) spatial structure on the horizontal plane was observed in all layers. Only 45% of the field showed a marked effect of tillage on PR. Mapping the effect of tillage on PR would be a useful approach for evaluating the global and local response of soil to tillage, as well as for delineating of areas within field for site-specific tillage practices.     

Poultry manure and banana waste are effective biofertilizer carriers for promoting plant growth and soil sustainability in banana crops

The aims of our study were to compare the effectiveness of poultry manure (PM) and banana waste (BW), with regard to their use as inoculant carriers of a bacterial consortium constituted by strains of Azospirillum, Azotobacter and P-solubiliser bacteria and to establish the most efficient dose of biofertilizer for a soil cultivated with banana (Musa paradisiaca AAA Simmonds), with respect to improving plant performance and soil physical and microbiological properties. Six months after planting, plant growth had increased with increase in dose of the biofertilizers applied. The biofertilizer prepared on BW enhanced the density of P-solubiliser bacteria, the concentrations of available P and foliar P to a greater extent than with the biofertilizer prepared on PM. The increases produced by the biofertilizer prepared on PM for the soil aggregate stability, enzymatic activities and the labile carbon fractions were highly correlated to the dose applied. Both biofertilizers can be considered potentially useful as inoculant carriers of PGPR but the usefulness of BW appears to be restricted to moderate doses of application (≤3%).