Co-occurrence and genotypic distribution of Phytophthora species recovered from watersheds and plant nurseries of eastern Tennessee

In 2008 statewide surveys of symptomatic foliage of nursery plants from Tennessee resulted in isolation of 43 isolates of Phytophthora spp. This sample set includes four described species (P. citrophthora, P. citricola, P. nicotianae, P. syringae), and a provisional species of Phytophthora ('P. hydropathica'). At the same time a stream-baiting survey was initiated to recover Phytophthora from eight watersheds in eastern Tennessee, some of which are near plant nurseries. Baiting was accomplished by submerging healthy Rhododendron leaves approximately 1 wk and isolation onto selective media. Six baiting periods were completed, and in total 98 Phytophthora isolates and 45 isolates of Pythium spp. were recovered. Three described species (P. citrophthora, P. citricola and P. irrigata) and the provisional species 'P. hydropathica' were obtained as well as three undescribed Phytophthora taxa and Pythium litorale. Isolates from both surveys were identified to species with morphology and the internal transcribed spacer (ITS) sequence. Isolates from species co-occurring in streams and nurseries (P. citricola, P. citrophthora and 'P. hydropathica') were characterized further with amplified fragment length polymorphism (AFLP) analyses and mefenoxam tolerance assays. Isolates representing a putative clonal genotype of P. citricola were obtained from both environmental and nursery sample sets.     

Rapid genotyping of the osteoporosis-associated polymorphic transcription factor Sp1 binding site in the COL1A1 gene by pyrosequencing

We describe a novel polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) sequencingbased assay for rapid genotyping of the polymorphic Sp1 binding site in the COL1A1 gene (1). A single nucleotide G-->T substitution polymorphism at this GC-rich site has recently been reported to be a predictive genetic marker for low bone mineral density (BMD). To simplify screening for this marker, we optimized PCR conditions and subjected the amplicons to pyrosequencing, which is a convenient high-throughput sequence analysis technique, readily amenable to automation. The analysis of 200 deidentified convenience DNA samples extracted from blood revealed genotype frequences in Hardy-Weinberg equilibrium (SS 68.0%, Ss 28.5%, and ss 3.5%) in agreement with other studies of European populations. This study demonstrates for the first time that pyrosequencing can be used for rapid identification of the osteoporosis-associated single nucleotide polymorphism (SNP) in the COL1A1 gene.     

Public Support for Conserving Bird Species Runs Counter to Climate Change Impacts on Their Distributions

There is increasing evidence that global climate change will alter the spatiotemporal occurrences and abundances of many species at continental scales. This will have implications for efficient conservation of biodiversity. We investigate if the general public in Denmark are willing to pay for the preservation of birds potentially immigrating and establishing breeding populations due to climate change to the same extent that they are for native species populations currently breeding in Denmark, but potentially emigrating due to climate change. We find that Danish citizens are willing to pay much more for the conservation of birds currently native to Denmark, than for bird species moving into the country – even when they are informed about the potential range shifts associated with climate change. The only exception is when immigrating species populations are under pressure at European level. Furthermore, people believing climate change to be man-made and people more knowledgeable about birds tended to have higher WTP for conservation of native species, relative to other people, whereas their preferences for conserving immigrant species generally resembled those of other people. Conservation investments rely heavily on public funding and hence on public support. Our results suggest that cross-country coordination of conservation efforts under climate change will be challenging in terms of achieving an appropriate balance between cost-effectiveness in adaptation and the concerns of a general public who seem mostly worried about protecting currently-native species.     

Over-expression of peptide deformylase in chloroplasts confers actinonin resistance,but is not a suitable selective marker system for plastid transformation

Arabidopsis thaliana peptide deformylase PDF1B was expressed in tobacco chloroplasts using spectinomycin as the selective agent. The foreign protein accumulated in chloroplasts (6% of the total soluble protein) and was enzymatically active. Transplastomic plants were evaluated for resistance to the peptide deformylase inhibitor actinonin. In vitro seed germination in the presence of actinonin and in planta application of the inhibitor demonstrated the resistance of the transformed plants. In addition, transgenic leaf explants were able to develop shoots via organogenesis in the presence of actinonin. However, when the combination of the PDF1B gene and actinonin was used as the primary selective marker system for chloroplast transformation of tobacco, all developed shoots were escapes. Therefore, under the experimental conditions tested, the use of this system for plastid transformation would be limited to function as a secondary selective marker.     

Replication initiation at the Escherichia coli chromosomal origin

To initiate DNA replication, DnaA recognizes and binds to specific sequences within the Escherichia coli chromosomal origin (oriC), and then unwinds a region within oriC. Next, DnaA interacts with DnaB helicase in loading the DnaB-DnaC complex on each separated strand. Primer formation by primase (DnaG) induces the dissociation of DnaC from DnaB, which involves the hydrolysis of ATP bound to DnaC. Recent evidence indicates that DnaC acts as a checkpoint in the transition from initiation to the elongation stage of DNA replication. Freed from DnaC, DnaB helicase unwinds the parental duplex DNA while interacting the cellular replicase, DNA polymerase III holoenzyme, and primase as it intermittently forms primers that are extended by the replicase in duplicating the chromosome.     

Larval and juvenile Pacific herring Clupea pallasii are not susceptible to infectious hematopoietic necrosis under laboratory conditions

Infectious hematopoietic necrosis (IHN) leads to periodic epidemics among certain wild and farmed fish species of the Northeast (NE) Pacific. The source of the IHN virus (IHNV) that initiates these outbreaks remains unknown; however, a leading hypothesis involves viral persistence in marine host species such as Pacific herring Clupea pallasii. Under laboratory conditions we exposed specific pathogen-free (SPF) larval and juvenile Pacific herring to 10(3) to 10(4) plaque-forming units (pfu) of IHNV ml(-1) by waterborne immersion. Cumulative mortalities among exposed groups were not significantly different from those of negative control groups. After waterborne exposure, IHNV was transiently recovered from the tissues of larvae but absent in tissues of juveniles. Additionally, no evidence of viral shedding was detected in the tank water containing exposed juveniles. After intraperitoneal (IP) injection of IHNV in juvenile herring with 10(3) pfu, IHNV was recovered from the tissues of sub-sampled individuals for only the first 5 d post-exposure. The lack of susceptibility to overt disease and transient levels of IHNV in the tissues of exposed fish indicate that Pacific herring do not likely serve a major epizootiological role in perpetuation of IHNV among free-ranging sockeye salmon Oncorhynchus nerka and farmed Atlantic salmon Salmo salar in the NE Pacific.     

Legacy microsite effect on the survival of bitterbrush outplantings after prescribed fire: capitalizing on spatial variability to improve restoration

Restoration of shrubs in arid and semi‐arid rangelands is hampered by low success rates. Planting shrub seedlings is a method used to improve success in these rangelands; however, it is expensive and labor intensive. The efficiency of shrub restoration could be improved by identifying microsites where shrub survival is greater. Bitterbrush (Purshia tridentata Pursh DC) is an important shrub to wildlife that has declined because of conifer encroachment, excessive defoliation, wildfires, and low recruitment. We investigated planting bitterbrush seedlings in western juniper (Juniperus occidentalis ssp. occidentalis Hook) encroached shrublands after prescribed fire was used to control trees. Bitterbrush seedlings were planted in under (canopy) and between (interspace) former juniper canopies at five blocks and evaluated for three growing seasons. Bitterbrush survival was greater than 50% in the former canopy, but only 5% in the interspace microsite by the third growing season. Growth of bitterbrush was also greater in the former canopy compared with the interspace, potentially due to markedly less perennial vegetation in this microsite. Exotic annual grasses and annual forbs became prevalent in the former canopy in the second and third growing season, suggesting that soil resource availability was greater in this microsite. These results suggest that restoration success will vary by specific locations within a burned landscape and that this variability can be used to improve restoration efficiency. In this situation, bitterbrush restoration can be improved by planting seedlings in former canopy compared with interspace microsites.     

Quantitative phosphoproteome analysis of lysophosphatidic acid induced chemotaxis applying dual-step (18)O labeling coupled with immobilized metal-ion affinity chromatography

Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in various cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its application for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed (16)O/ (18)O labeling plus (16)O/ (18)O-methanol esterification for quantitation, a macro-immobilized metal-ion affinity chromatography trap for phosphopeptide enrichment, and LC-MS/MS analysis. LC separation and MS/MS are followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer. A variety of phosphorylated proteins were identified and quantified including receptors, kinases, proteins associated with small GTPases, and cytoskeleton proteins. A number of hypothetical proteins were also identified as differentially expressed followed by LPA stimulation, and we have shown evidence of pseudopodia subcellular localization of one of these candidate proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with LPA gradient sensing and cell chemotaxis.