DNA markers provide insight about common lime in historical plantings – An example from the Royal Danish Gardens

As part of the restoration process of an avenue of common lime (Tilia × europaea) from 1760 in the Royal Danish Gardens, all remaining trees were genotyped with DNA markers before they were felled. As such, information about the nature of the plant material (clonal versus non-clonal) and mode of propagation was obtained, revealing that a single clone constituted 92% of the remaining trees (106 out of 115). Five trees were of another clone, while the remaining four trees had unique genotypes. Mode of clonal propagation was most likely layering since the genotype of the crown and the roots of a subsample of the trees had the same genotype. Trees from four other locations with historical avenues/plantings from the 17th century were also genotyped. The two clones registered in the first location were also found at the other four locations. Of 76 trees from the other historical avenues/plantings, only two trees did not belong to either of the two clones. Genotyping of commercial common lime trees that would be planted in place of the felled trees during the restoration project was also performed. Samples of 20 newly planted trees all possessed the same genotype as the majority of the old felled trees and, thereby, were the same clone as the trees planted nearly 250 years ago. Altogether, the current study shows that the genetic diversity of common lime planted in Danish historical plantings is extremely narrow, and that the same clones have been produced for decades/centuries by private nurseries in the Netherlands and Germany. It also provides evidence that it is possible to obtain the same genetic material as originally planted when common lime trees are to be replaced in historical plantings. Furthermore, the utility of DNA markers in the management of plant material in parks is demonstrated.     

Isolation and Differentiation of Mesenchymal Stem Cells From Bovine Umbilical Cord Blood

Currently, mesenchymal stem cells (MSCs) are used in veterinary clinical applications. Bone marrow and adipose tissue are the most common sources of stem cells derived from adult animals. However, cord blood which is collected non‐invasively is an alternative source of stem cells other than bone marrow and adipose tissue. Moreover, high availability and lower immunogenicity of umbilical cord blood (UCB) haematopoietic stem cells compared to other sources of stem cell therapy such as bone marrow have made them a considerable source for cell therapy, but MSCs is not highly available in cord blood and their immunogenicity is poorly understood. In this study, the cells with spindle morphology from 7 of 9 bovine UCB samples were isolated and cultured. These mesenchymal stromal cells were successfully differentiated to osteocytes, chondrocytes and adipocytes. In addition, Oct‐4 and SH3 were determined by RT‐PCR assay. It is the first report of isolation, culture, characterization and differentiation of bovine umbilical stem cells.     

Fusarium head blight of cereals in Denmark: species complex and related mycotoxins

Quantitative real-time polymerase chain reaction differentiating 10 Fusarium spp. and Microdochium nivale or M. majus was applied to a total of 396 grain samples of wheat, barley, triticale, oat, and rye sampled across Denmark from 2003 to 2007, along with selected samples of wheat and barley from 1957 to 2000, to determine incidence and abundance of individual Fusarium spp. The mycotoxins deoxynivalenol (DON), nivalenol, zearalenone, T-2, and HT-2 were quantified using liquid chromatography-double mass spectrometry. Major differences in the Fusarium species complex among the five cereals as well as great yearly variation were seen. Fusarium graminearum, F. culmorum, and F. avenaceum were dominant in wheat, with DON as the dominant mycotoxin. F. langsethiae, F. culmorum, and F. avenaceum were dominant in barley and oat, leading to relatively high levels of the mycotoxins T-2 and HT-2. F. graminearum, F. culmorum, and F. avenaceum dominated in triticale and rye. The nontoxigenic M. nivale/majus were present in significant amounts in all cereal species. Wheat and barley samples from 1957 to 1996 exhibited no or very low amounts of F. graminearum, indicating a recent increase of this pathogen. Biomass and mycotoxin data exhibited good correlations between Fusarium spp. and their corresponding mycotoxins under field conditions.     

Pioneer 11 observations of energetic particles in the jovian magnetosphere

Knowledge of the positional distributions, absolute intensities, energy spectra, and angular distributions of energetic electrons and protons in the Jovian magnetosphere has been considerably advanced by the planetary flyby of Pioneer 11 in November-December 1974 along a quite different trajectory from that of Pioneer 10 a year earlier. (i) The previously reported magnetodisc is shown to be blunted and much more extended in latitude on the sunward side than on the dawn side. (ii) Rigid corotation of the population of protons E(p) approximately 1 million electron volts in the magnetodisc is confirmed. (iii) Angular distributions of energetic electrons E(e) > 21 million electron volts in the inner magnetosphere are shown to be compatible with the Kennel-Petschek whistler-mode instability. (iv) A diverse body of magnetospheric effects by the Jovian satellites is found. (v) Observations of energetic electrons in to a radial distance of 1.59 Jovian radii provide a fresh basis for the interpretation of decimetric radio noise emission.     

Catch Crop and Animal Slurry in Spring Barley Grown with Straw Incorporation

Abstract

Four rates of straw (0, 4, 8 and 12 t ha^?1 yr^?1) were incorporated in a field experiment with continuous spring barley. The experiment was conducted on a sandy soil (5.5% clay) and a sandy loam soil (11.2% clay). After eight years, the straw incorporation was combined with catch-crop growing with and without winter application of animal slurry and also spring fertilization with mineral fertilizer (0, 50, 100 or 125 kg N ha^?1 yr^?1). The combined experiment was conducted for three lyears on the sandy soil and for four years on the sandy loam soil. The effects on barley dry matter yield and N uptake are presented together with the long-term effects of the straw incorporations on crop growth and soil C and N. Grain yield on the sandy loam was unaffected by straw incorporation. On the sandy soil the highest straw application rates reduced grain yield in the unfertilized barley. When the barley received mineral fertilizer at recommended levels (100 kg N ha^?1 yr^?1), grain yield on this soil was also unaffected by the high straw rates. Including a catch crop had a positive effect on the grain yield of barley on both soils. The total N uptake in grain and straw generally increased with straw application up to 8 t ha^?1 yr^?1. With the highest straw application rate (12 t ha^?1 yr^?1), the total N uptake decreased but still exceeded N uptake in barley grown with straw removal. The barley accumulated higher amounts of N when a catch crop was included. The total N uptake in the barley was significantly higher after animal slurry application. The extra N uptake, however, was much lower than the amounts of N applied with the slurry. Incorporation of straw had only a small influence on N uptake after slurry application. The straw, therefore, was not able to store the applied N during winter. In the two four-year periods before the combined experiment, grain yield on the sandy loam was generally negatively affected by straw incorporations. In the second period, N uptake began to show a positive effect of the straw. On the sandy soil, grain yield and N uptake during the whole period were generally positively affected by the straw incorporations except for the highest straw rate (12 t ha^?1 yr^?1). The sandy loam soil showed higher increases in C and N content after the repeated straw incorporations and catch-crop growing than the sandy soil. When application of animal slurry was combined with the catch crop, no further increases in soil C and N were found relative to soil where a catch crop was grown without slurry application. Large amounts of the N applied with the slurry may therefore have been lost by denitrification or nitrate leaching.     

Dead or alive — Old empty shells do not prompt false-positive results in environmental DNA surveys targeting the freshwater pearl mussel (Margaritifera margaritifera L.)

1. Environmental DNA (eDNA) from water samples is increasingly used to detect the presence and distribution of species in aquatic ecosystems. However, before implementing eDNA in monitoring programmes, various species-specific sampling or analytical issues remain to be resolved in order to minimize frequencies of false-positive and -negative results. For example, empty shells from freshwater pearl mussels (Margaritifera margaritifera) contain extractable DNA (chemical extraction from ground-up shells) suggesting a risk of false-positive samples at stream sites with extinct populations but with empty shell material remaining.
2. The aim of this study was to investigate whether empty and naturally degrading shells from M. margaritifera can cause false-positive eDNA signals in water samples.
3. Water samples were collected from outdoor stream channels (in Lemming, Denmark) with living freshwater pearl mussels or empty shell material (density ~10 individuals m⁻²) during a 3-week experimental period. Living freshwater pearl mussels were collected from Hemgravs stream in Sweden and transported to Denmark according to permissions granted by the Swedish and Danish authorities.
4. All water samples from stream channels containing empty shells were negative for eDNA indicating that eDNA traces in stream water are most likely to originate from living individuals located upstream of the sampling site. Water samples collected from stream channels containing living individuals of M. margaritifera were consistently positive for eDNA except for one sample (interpreted as a false negative).
5. The study shows that positive eDNA signals for freshwater pearl mussels most likely reflect the presence of living individuals. Consequently, we suggest that eDNA should be used to locate remaining population fragments of M. margaritifera in deep and turbulent streams, providing a platform for faster and more efficient decision making when launching investigative and mitigation initiatives.

     

Phenotypic and genotypic characterisation of Pasteurella multocida isolated from pigs at slaughter in New Zealand

AIMS: To examine pigs at slaughter in New Zealand for the presence of Pasteurella multocida, and to determine for isolates, their biochemical profiles, somatic and capsular types, and the presence or absence of the HSB and toxA genes, associated with haemorrhagic septicaemia (HS) and progressive atrophic rhinitis (PAR), respectively.

METHODS: Swabs from 173 lungs, 158 palatine tonsils and 82 nasal passages of pigs at two abattoirs in New Zealand were cultured for P. multocida using conventional techniques, and isolated colonies were subjected to biochemical tests for identification of biovars. Somatic serotyping was conducted using an agar gel immunodiffusion (AGID) test. Polymerase chain reaction (PCR) assays were used to confirm phenotypic identification of colonies using species-specific primers, capsule type using serogroup-specific primers and multiplex PCR, and to test for the presence of HSB and toxA genes.

RESULTS: Pasteurella multocida was isolated from 11/173 (6.4%) lung, 32/158 (20.2%) palatine tonsil and 5/82 (6.1 %) nasal swab samples, a total of 48 isolates from 413 samples (11.6%). Isolation rates per farm ranged from 1–53% of tissue samples collected from pigs 5–6 months of age. On phenotypic characterisation, isolates were allocated to seven main biovars, viz 1, 2, 3, 5, 9, 12, and a dulcitol-negative variant of Biovar 8, the majority (30/48) being Biovar 3. Of the 42 isolates for which somatic serotyping was conducted, 10% were Serovar 1, 79% were Serovar 3, 2% were Serovar 6,1, 2% were Serovar 12, and 7% could not be typed. All 48 isolates were confirned as P. multocida using a species-specific PCR. In the capsular multiplex PCR, 92% of isolates were Capsular (Cap) type A, 2% were Cap D, and 6% could not be typed. None of the samples were positive for the HSB or toxA genes.

CONCLUSION: Serovars or capsular types of P. multocida associated with HS or PAR in pigs were not detected. Establishment of species-specific, capsular and toxin PCR assays allowed the rapid screening of isolates of P. multocida, while serotyping provided an additional tool for epidemiological and tracing purposes.     

Detection of Mycoplasma conjunctivae in sheep affected with conjunctivitis and infectious keratoconjunctivitis

AIM: To determine the aetiolog y of a recurring and severe form of infectious keratoconjunctivitis (IKC) in sheep.

METHODS: Five sheep flocks that had experienced a severe form of IKC were examined. Clinical history, conjunctival swabs and blood samples were collected from affected animals. Culture for bacteria, and also specifically for Mycoplasma and Chlamydophila spp, and detection of Mycoplasma conjunctivae DNA by polymerase chain reaction (PCR) were attempted. Serum samples were tested for antibodies to M. agalactiae, M. capricolum, M. conjunctivae and Chlamydophila spp.

RESULTS: Mycoplasma conjunctivae DNA was detected using PCR in 3/5 flocks, and in all flocks antibodies to M. conjunctivae were detected in sera. A pure growth of Branhamella ovis was cultured from conjunctival swabs from a small proportion of sheep in two flocks. No other pathogens were detected.

CONCLUSIONS: This investigation demonstrated that M. conjunctivae was a primary pathogen causing severe IKC in sheep, and is the first report of detection of this organism in sheep in New Zealand. Introduction of clinically normal carrier sheep appeared to have caused the outbreaks.