Two-dimensional single strand conformation polymorphism (SSCP) of 16S rRNA gene fragments reveals highly dissimilar bacterial communities in an acidic fen

Genetic fingerprinting methods such as denaturing gradient gel electrophoresis (DGGE) and single strand conformation polymorphism (SSCP) are only able to separate about 20–40 well-distinguishable bands (signals) within each sample. As a result, the diversity of 16S rRNA genes within biological samples may be underestimated, because multiple sequences can migrate at the same rate to form a single band. This study reports a two-dimensional SSCP fingerprinting method that has the capability to resolve hundreds of signals in a single fingerprint by using different gel temperatures in the two dimensions of the separation (20 °C and 30 °C, respectively). Unlike previous two-dimensional approaches, the method presented in this study does not rely on DNA products of variable lengths but is able to separate 16S rRNA gene fragments of the same length. To demonstrate the effectiveness of this new method, DNA samples from oxic and anoxic zones of an acidic fen were examined. Whereas one-dimensional SSCP fingerprints indicated high similarity (>93%) between 16S rRNA gene fragments from oxic and anoxic zones of the fen, the two-dimensional SSCP approach virtually found no similarities.     

Efficacy of Clausena anisata and Plectranthus glandulosus leaf powder against Prostephanus truncatus (Coleoptera: Bostrichidae) and two strains of Sitophilus zeamais (Coleoptera: Curculionidae) on maize

Powdered leaves of Clausena anisata and Plectranthus glandulosus were evaluated on Cameroonian and German strains of Sitophilus zeamais and Prostephanus truncatus for their insecticidal activities, the effects on progeny production, grain damage and population increase by admixing powders and maize grains. The two powders caused significant mortality of the three insects with P. glandulosus being far more effective than C. anisata. P. truncatus was less susceptible to P. glandulosus powder compared with the two strains of S. zeamais. The German strain (LD₅₀ = 2.75 g/100 g grain) of S. zeamais was less susceptible to P. glandulosus powder than the Cameroonian strain (LD₅₀ = 1.86 g/100 g grain). P. glandulosus unlike C. anisata powder, considerably reduced grain damage, progeny production and population increase. This study shows that there is a great potential for the powder of P. glandulosus as a promising grain protectant for integration within the subsistence farming systems of sub-saharan Africa.     

Length curves and volume functions for guadua bamboo (Guadua angustifolia Kunth) for the coffee region of Colombia

Three models for the prediction of bamboo culm length and two for culm volume were fitted from data of 303 guadua bamboo (Guadua angustifolia Kunth) culms. Data are from 101 temporary inventory plots systematically distributed over the coffee region of Colombia (inventory area about 1,029,525 ha). Linear and non-linear regression models were used, and the precision of the models was evaluated by cross-validation. The models were compared by studying the adjusted coefficient of determination, the bias, mean square error and Akaike’s information criterion and by the F-test that compares predicted and observed values. For culm length, the best fit showed models that included predictor variables related to stand characteristics such as quadratic mean diameter and number of culms per ha. For culm volume prediction, the inclusion of culm length improved the model significantly. For the simple form factor model, precision of prediction was least. The models developed are useful in facilitating the estimation of stand characteristics that are relevant for the silvicultural management of Guadua stands and also for the assessment of their environmental services (such as carbon sequestration).     

Temporal variations of crop residue effects on soil N transformation depend on soil properties as well as residue qualities

Purple soils (Eutric Regosols) are widely distributed in humid subtropical Southwest China. They are characterized by high nitrification activities, with risks of severe NO₃^? leaching. Incorporation of crop residues is considered an effective method to reduce NO₃^? loss. In the present study, we compared the effects of alfalfa, rice straw, and sugarcane bagasse on gross N transformation turnover in a purple soil (purple soil, pH 7.62) compared with those in an acid soil (acid soil, pH 5.26), at 12 h, 3 months, and 6 months after residue incorporation. The gross N transformation rates were determined by ¹⁵N tracing. All tested crop residues stimulated the gross N mineralization rates, but reduced the net mineralization rates in both soils at 12 h after residue incorporation; however, the extent of the effect varied with the crop residue qualities, with rice straw having the strongest effects. Crop residues reduced net nitrification rates by depressing gross autotrophic nitrification rates and stimulating NO₃^? immobilization rates in the purple soil, particularly after rice straw incorporation (net nitrification rate decreased from 16.72 mg N kg^?1 d^?1 in the control to ??29.42 mg N kg^?1 d^?1 at 12 h of residue incorporation); however, crop residues did not affect the gross autotrophic nitrification rates in the acid soil. Crop residue effects subsided almost completely within 6 months, with sugarcane bagasse showing the longest lasting effects. The results indicated that crop residues affected the N transformation rates in a temporal manner, dependent on soil properties and residue qualities.     

Bacterivore nematodes stimulate soil gross N transformation rates depending on their species

We conducted a microcosm experiment with soil being sterilized, reinoculated with native microbial community and subsequently manipulated the bacterivorous nematodes, including three treatments: without (CK) or with introducing one species of the two bacterivores characterized with different body size but similar c-p (colonizer-persister) value (Rhabditis intermedia and Protorhabditis oxyuroides, accounted for 6 and 59% of bacterivores in initially undisturbed soil, respectively). We monitored the N₂O and CO₂ emissions, soil properties, and especially quantified gross N transformation rates using ¹⁵N tracing technique after the 50 days incubation. No significant differences were observed on soil NH₄ ⁺ and NO₃ ^? concentrations between the CK and two bacterivores, but this was not the case for gross N transformation rates. In comparison to CK, R. intermedia did not affect soil N transformation rates, while P. oxyuroides significantly increased the rates of mineralization of organic N to NH₄ ⁺, oxidation of NH₄ ⁺ to NO₃ ^?, immobilization of NO₃ ^? to organic N and dissimilatory NO₃ ^? reduction to NH₄ ⁺. Furthermore, the mean residence time of NH₄ ⁺ and NO₃ ^? pool was greatly lowered by P. oxyuroides, suggesting it stimulated soil N turnover. Such stimulatory effect was unrelated to the changes in abundance of bacteria and ammonia-oxidizing bacteria (AOB). In contrast to CK, only P. oxyuroides significantly promoted soil N₂O and CO₂ emissions. Noticeably, bacterivores increased the mineralization of recalcitrant organic N but decreased soil δ¹³C_-TOC and δ¹⁵N_-TN values, in particular for P. oxyuroides. Combining trait-based approach and isotope-based analysis showed high potential in moving forward to a mechanistic understanding of bacterivore-mediated N cycling.     

Stimulation of methane oxidation by CH<Subscript>4</Subscript>-emitting rose chafer larvae in well-aerated grassland soil

In this study, the impact of rose chafer (Cetonia aurata L.) larvae on net and gross methane (CH₄) fluxes in soil from an old permanent grassland site (Giessen, Germany) was investigated. Previous studies at this site suggested the existence of Scarabaeidae larvae-induced “CH₄-emitting hot spots” within the soil profile which may subsequently lead to increased CH₄ oxidation. The net (soil + larvae) and gross (soil and larvae separated) CH₄ fluxes were studied in a 3-month laboratory incubation. Addition of larvae changed the soil from a net sink (?330 ± 11 ng CH₄ kg^?1 h^?1) to a net source (637 ± 205 ng CH₄ kg^?1 h^?1). Supply of plant litter to the soil + larvae incubation jars tended to increase CH₄ emissions which was not significant due to large variability. After 11–13 weeks of incubation, the net soil CH₄ oxidation was significantly stimulated by 13–21% in the treatments containing larvae when these were taken out. Analysis of archaeal 16S rRNA genes revealed that the majority of the obtained clones were closely related to uncultured methanogens from guts of insects and other animals. Other sequences were relative to cultivated species of Methanobrevibacter, Methanoculleus, and Methanosarcina. Hence, Scarabaeidae larvae in soils (i) may represent an underestimated source of CH₄ emissions in aerobic upland soils, (ii) may stimulate gross CH₄ consumption in their direct soil environment, and, thus, (iii) contribute to the spatial heterogeneity often observed in the field with closed-chamber measurements. Long-term CH₄-flux balances may be wrongly assessed when “exceptional” net CH₄ flux rates (due to larvae hot spots) are excluded from data sets.     

Evaluation of skin sensitivity after shock wave treatment in horses

OBJECTIVE: To evaluate the effects of shock wave treatment on cutaneous nerve function, compared with the effects of local nerve block and sedation. ANIMALS: 18 clinically sound Swiss Warmbloods. PROCEDURE: Horses were randomly allocated to 3 groups and received different amounts and types of shock waves (extracorporeal shock wave treatment [ESWT] or radial pressure wave treatment [RPWT]). Horses were sedated with xylazine and levomethadone. Shock waves were applied to the lateral palmar digital nerve at the level of the proximal sesamoid bones on 1 forelimb. Skin sensitivity was evaluated by means of an electrical stimulus at the coronary band before and 5 minutes after sedation and at 4, 24, and 48 hours after application of ESWT or RPWT. On the contralateral forelimb, skin sensitivity was tested before and 10 minutes after an abaxial sesamoid nerve block. RESULTS: No significant changes in skin sensitivity were detected, regardless of the shock wave protocol applied. Mean reaction thresholds after sedation were more than twice the baseline thresholds. After the abaxial sesamoid block, no reaction was recorded in any of the horses. CONCLUSIONS AND CLINICAL RELEVANCE: Application of ESWT or RPWT to the palmar digital nerve had no effect on cutaneous sensation distal to the treated region for at least 2 days after application. The analgesic effect of sedation on reaction to electrical stimuli was distinct but varied among horses.     

Insektizide und phytotoxische Wirkung von Steinkohlenflugasche

Zusammenfassung  Im Rahmen eines Umweltgutachtens wurde die phytotoxische und insektizide Wirkung von Steinkohlenflugasche untersucht. Als Testorganismen dienten die Pflanzen Tropaeolum majus (Kapuzinerkresse) und Lepidium sativum (Brunnenkresse) sowie die Vorratsschädlinge Plodia interpunctella (Dörrobstmotte), Tenebrio molitor (Mehlkäfer) und Sitophilus granarius (Kornkäfer). Gezeigt werden konnte, dass die verwendete Steinkohlenflugasche bei einem aus der Literatur entnommenen theoretischen täglichen Flugstaubniederschlag von 1 g/m² keine phytotoxischen bzw. insektiziden Eigenschaften aufwies. Selbst eine zehnfach erhöhte Dosis von 10 g/m² hatte innerhalb von 2 Tagen keine erhöhte Mortalität von Dörrobstmottenlarven bzw. über einen Zeitraum von 12 Tagen keine erhöhte Mortalität von Mehlkäfern (alle Entwicklungsstadien) zur Folge. Für Kornkäfer ergab sich eine leichte Erhöhung der Mortalität auf 2 % innerhalb von 12 Tagen nach einer Dosierung von 5 g/m². Die Mortalitätsrate von Kornkäfern erhöhte sich nach Applikation von 125 mg Flugasche auf eine Fläche von 50 cm² innerhalb von 12 Tagen von 6 % in der Kontrolle auf 14 %. Eine mit Diatomeenerde zur Positivkontrolle durchgeführte Versuchsreihe hatte hingegen bei gleich hoher Dosierung eine 100 %ige Abtötung aller adulten Kornkäfer zur Folge. Starke Pflanzausfälle waren erst bei einem komplett aus Flugasche bestehendem Substrat zu verzeichnen. Nur geringfügig waren die Wuchsleistungen der verwendeten Pflanzen ab einem Flugascheanteil von 50 % im Substrat beeinträchtigt.

Development of two real-time PCR assays for the detection of Mycoplasma hyopneumoniae in clinical samples

In order to improve the diagnosis of enzootic pneumonia (EP) in pigs two real-time polymerase chain reaction (rtPCR) assays for the detection of Mycoplasma hyopneumoniae in bronchial swabs from lung necropsies were established and validated in parallel. As a gold standard, the current "mosaic diagnosis" was taken, including epidemiological tracing, clinical signs, macro- and histopathological lesions of the lungs and immunofluorescence. One rtPCR is targeting a repeated DNA element of the M. hyopneumoniae genome (REP assay), the other a putative ABC transporter gene (ABC assay). Both assays were shown to be specific for M. hyopneumoniae and did not cross react with other bacteria and mollicutes from pig. With material from pigs of defined EP-negative farms the two assays showed to be 100% specific. When testing lungs from pig farms with EP, the REP assay detected 50% and the ABC assay 90% of the farms as positive. Both tests together detected all positive farms. Within a positive herd the two assays tested similarly with on average over 90% of the lung samples analysed from a single farm showing positive scores. A series of samples with suspicion of EP and samples from pigs with diseases other than respiratory taken from current routine diagnostic was assayed. None of the assays showed false positive results. The sensitivities in this sample group were 50% for the REP and 70% for the ABC assays and for both assays together 85%. The two assays run in parallel are therefore a valuable tool for the improvement of the current diagnosis of EP.