Quantitative real-time PCR effectively detects and quantifies colonization of sclerotia of Sclerotinia sclerotiorum by Trichoderma spp.

Some members of the fungal genus Trichoderma are able to colonize and destroy sclerotia, the thick-walled resting structures of the soilborne plant pathogenic fungus Sclerotinia sclerotiorum, thus providing a potential means of biological disease control. However, current methods to detect and quantify colonization of sclerotia are labor-intensive, and generally qualitative rather than quantitative in nature. Our objective was to develop quantitative real-time PCR (polymerase chain reaction) methods to detect and measure colonization of sclerotia by Trichoderma spp. Specific PCR primer/probe sets were developed for Trichoderma spp. and for S. sclerotiorum. A total of 180 ITS1 (internal transcribed spacer) and ITS2 sequences from different species in the genus Trichoderma were aligned, and consensus sequences were determined. Six candidate primer sets were based on conserved regions of the consensus sequence, and the specificity of each nucleotide sequence was examined using BLAST (Basic Local Alignment Search Tool; NCBI) software. Each candidate primer set was tested on genomic DNA of T. harzianum strain ThzID1-M3, as well as six different Trichoderma isolates from soil, and on genomic DNA of S. sclerotiorum. The optimum primer/probe set selected, TGP4, successfully amplified genomic DNA of all Trichoderma isolates tested, and showed high precision and reproducibility over a linear range of eight orders of magnitude, from 85 ng to 8.5 fg of T. harzianum genomic DNA. TGP4 did not amplify S. sclerotiorum DNA. A specific PCR primer/probe set (TMSCL2) was developed for S. sclerotiorum, based on the calmodulin gene sequence. TMSCL2 did not amplify Trichoderma DNA. Quantitative real-time PCR with the primers then was evaluated in experiments to test differential effects of two soil moisture levels (−50 kPa, −1000 kPa matric potential) on biocontrol of S. sclerotiorum by indigenous Trichoderma spp. Periodically over 40 days, Trichoderma and S. sclerotiorum DNA in sclerotia were quantified by PCR with appropriate primers. Over 90% of the sclerotia were colonized by indigenous Trichoderma spp. at −1000 kPa, over the 40-day period, compared to only 60% at −50 kPa. In addition to determining incidence of colonization, the PCR method allowed measurement of the extent of sclerotial colonization, which also was significantly greater in the drier soil. Quantitative real-time PCR with the TGP4 primer/probe set provides a sensitive detection and measurement tool to evaluate colonization of sclerotia by Trichoderma spp.     

Isolation of border disease virus from twin lambs in Alberta

We describe herein a field case of border disease (BD) in twin lambs. Both lambs were unthrifty, stunted, and one exhibited nervous signs characteristic of BD, with tremors of the head, neck, hind legs, and pelvis. Hairiness of the coat and excessive pigmentation, often seen in lambs with BD, were not observed. A noncytopathic virus, which showed cross-reactivity with bovine viral diarrhea (BVD) virus antiserum and BVD virus monoclonal antibodies, was isolated repeatedly from leukocytes from one lamb and from tissues of the other. Although the source of the virus is unknown, our results suggest that the dam of the affected twins had been infected during pregnancy. We used the BD virus isolated to inoculate pregnant ewes and experimentally reproduce the disease in a newborn lamb. Our findings indicate that leukocytes, rather than serum, should be utilized for BD virus isolation. Further, it is recommended that BD virus, rather than BVD virus, be used in serum neutralization tests when screening sheep for antibody titers.     

Saponin-containing subfractions of soybean molasses induce enteritis in the distal intestine of Atlantic salmon

The current work aimed at tracing the causative components for soybean-induced enteritis in Atlantic salmon (Salmo salar L.). Soybean molasses was subjected to phase separation using n-butanol. Three subfractions were obtained as follows: butanol phase, precipitate, and water phase. The biochemical composition of soybean molasses and the obtained subfractions were analyzed in detail: Protein, fat, and ash were quantified according to standard methods. Sucrose, raffinose, and stachyose were quantified using high-performance anion-exchange chromatography. Soyasaponins were quantified using reverse-phase high-performance liquid chromatography. Finally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to evaluate the size distribution of the proteins present in each fraction. Molasses and the different subfractions were thereafter fed to Atlantic salmon in two successive fish trials. The level of intestinal inflammation was evaluated by light microscopy using a semiquantitative scoring system. Histological assessments revealed that Atlantic salmon fed a combination of butanol phase and precipitate displayed significant enteritis. Atlantic salmon fed the water phase displayed normal intestinal morphology. The causative components for soybean-induced enteritis withstand butanol treatment and prolonged heating at 70 degrees C. Sucrose, raffinose, stachyose, nor soybean proteins larger than 10 kDa induce enteritis alone. Soyasaponins, or components that follow the same solubility pattern, trigger the inflammatory reaction. We therefore suggest that soybean-induced enteritis in Atlantic salmon is induced by soyasaponins alone or by soyasaponins in combination with other factors, e.g., antigenic soybean proteins or the intestinal microflora.     

Post-cambrian trilobite diversity and evolutionary faunas

A cluster analysis of the stratigraphic distribution of all Ordovician trilobite families, based on a comprehensive taxonomic database, identified two major faunas with disjunct temporal diversity trends. The Ibex Fauna behaved as a cohort, declining through the Ordovician and disappearing at the end-Ordovician mass extinction. In contrast, the Whiterock Fauna radiated rapidly during the Middle Ordovician and gave rise to all post-Ordovician trilobite diversity. Its pattern of diversification matches that of the Paleozoic Evolutionary Fauna; hence, trilobites were active participants in the great Ordovician radiations. Extinction patterns at the end of the Ordovician are related to clade size: Surviving trilobite families show higher genus diversity than extinguished families.     

Biogeochemical evidence for dinoflagellate ancestors in the early cambrian

Dinoflagellates are single-celled organisms that reflect the ecological conditions in modern oceans and lakes. Ultrastructural data and molecular phylogeny suggest that they originated in the Neoproterozoic, yet dinoflagellate ancestors are classified only to the Middle Triassic ( approximately 240 million years ago). Examination of dinoflagellate-specific biological markers (dinosteranes and 4alpha-methyl-24-ethylcholestane) in concentrated microfossils with known morphology identified ancient dinoflagellate ancestors from the Early Cambrian ( approximately 520 million years ago).     

Measuring the spin polarization of a metal with a superconducting point contact

A superconducting point contact is used to determine the spin polarization at the Fermi energy of several metals. Because the process of supercurrent conversion at a superconductor-metal interface (Andreev reflection) is limited by the minority spin population near the Fermi surface, the differential conductance of the point contact can reveal the spin polarization of the metal. This technique has been applied to a variety of metals where the spin polarization ranges from 35 to 90 percent: Ni0.8Fe0.2, Ni, Co, Fe, NiMnSb, La0.7Sr0.3MnO3, and CrO2.     

Visual instruction of the neural map of auditory space in the developing optic tectum

Neural maps of visual and auditory space are aligned in the adult optic tectum. In barn owls, this alignment of sensory maps was found to be controlled during ontogeny by visual instruction of the auditory spatial tuning of neurons. Large adaptive changes in auditory spatial tuning were induced by raising owls with displacing prisms mounted in spectacle frames in front of the eyes; neurons became tuned to sound source locations corresponding to their optically displaced, rather than their normal, visual receptive field locations. The results demonstrate that visual experience during development calibrates the tectal auditory space map in a site-specific manner, dictating its topography and alignment with the visual space map.     

Neptune's eccentricity and the nature of the kuiper belt

The small eccentricity of Neptune may be a direct consequence of apsidal wave interaction with the trans-Neptune population of debris called the Kuiper belt. The Kuiper belt is subject to resonant perturbations from Neptune, so that the transport of angular momentum by density waves can result in orbital evolution of Neptune as well as changes in the structure of the Kuiper belt. In particular, for a belt eroded out to the vicinity of Neptune's 2:1 resonance at about 48 astronomical units, Neptune's eccentricity can damp to its current value over the age of the solar system if the belt contains slightly more than an earth mass of material out to about 75 astronomical units.     

Individual feeding specialisation of a naïve vs. veteran predators

Abstract – We explored the incidence of individual feeding specialisation among a naïve predator (non‐native rainbow trout postsmolts) and two native experienced predators (sea‐run Arctic charr and sea‐trout) in a subarctic Norwegian fjord. Interindividual foraging niche stability was obtained by combining information on stomach contents (recent dietary niche) with trophically transmitted parasite infestation (time‐integrated historical dietary niche) of individual predators. Individual fish showed a high degree of resource specialisation as prey items such as gammarids and small fish (both potential intermediate host of parasites) rarely co‐occurred in stomachs. In both naïve and veteran predators, positive associations between the intensity of a specific parasite species and the occurrence of their respective intermediate host (gammarids or fish) in the stomachs of individual predators demonstrated temporally interindividual feeding specialisations. Several behavioural phenotypes clearly co‐existed in both naïve and veteran predator populations, including gammaridivore (benthic feeders), piscivore (pelagic feeders) or insectivore (pleuston feeders) individuals. The likely mechanism of this observed interindividual resource specialisation in the non‐native naïve predators involves a behavioural component of which rapid learning seems to be a key factor.     

The suitability of anadromous Arctic charr as host and vector of the monogenean Gyrodactylus salaris

Abstract –  In 2000, the Atlantic salmon ( Salmo salar L.) stock in the river Signaldalselva, North Norway was found to be infected with the monogenean Gyrodactylus salaris (Malmberg). This river system has a multispecies fish community of nine species in total. Low densities of Atlantic salmon parr were found during electrofishing in October (2003) with a prevalence of 94% and a mean abundance of 848 G. salaris. This watercourse also holds a riverine stock of anadromous Arctic charr ( Salvelinus alpinus ), which is not common in Norway. Among the parr of Arctic charr, 64% were infested with an abundance of 23 parasites per fish. The most heavily infected 0+ and 1+ juvenile Arctic charr had 469 and 534 parasites, respectively, indicating that the parasites are able to reproduce on Arctic charr in the wild. There was little variation in the rates of infestation of Arctic charr along the studied stretch of the river, even in areas with very low densities of Atlantic salmon parr. The parr of anadromous Arctic charr seem to be suitable as long-term hosts for G. salaris . Moreover, a few (22%) infested adult sea-running Arctic charr were captured after ascending the river in the autumn, which shows that large fish may also act as carriers of G. salaris . However, no parasites were recorded on potential anadromous Arctic charr before descending during early spring (April 2003 and April 2004). Thus, it is still not clear whether anadromous Arctic charr are able to disperse the parasite between watercourses. So far, it is uncertain whether G. salaris may increase the mortality rates of Arctic charr and thereby be a threat to these unusual occurring riverine anadromous Arctic charr stocks.