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Agroinfiltration in Nicotiana benthamiana is widely used to transiently express heterologous proteins in plants. However, the state of Agrobacterium itself is not well studied in agroinfiltrated tissues, despite frequent studies of immunity genes conducted through agroinfiltration. Here, we generated a bioluminescent strain of Agrobacterium tumefaciens GV3101 to monitor the luminescence of Agrobacterium during agroinfiltration. By integrating a single copy of the lux operon into the genome, we generated a stable 'AgroLux' strain, which is bioluminescent without affecting Agrobacterium growth in vitro and in planta. To illustrate its versatility, we used AgroLux to demonstrate that high light intensity post infiltration suppresses both Agrobacterium luminescence and protein expression. We also discovered that AgroLux can detect Avr/Cf-induced immune responses before tissue collapse, establishing a robust and rapid quantitative assay for the hypersensitive response (HR). Thus, AgroLux provides a non-destructive, versatile and easy-to-use imaging tool to monitor both Agrobacterium and plant responses.

Many host organisms live in polymicrobial environments and must respond to a diversity of pathogens. The degree to which host defences towards one pathogen species affect susceptibility to others is unclear. We used a panel of Caenorhabditis elegans nematode isolates to test for natural genetic variation in fitness costs of immune upregulation and pathogen damage, as well as for trade-offs in defence against two pathogen species, Staphylococcus aureus and Pseudomonas aeruginosa. We examined the fitness impacts of transient pathogen exposure (pathogen damage and immune upregulation) or exposure to heat-killed culture (immune upregulation only) by measuring host population sizes, which allowed us to simultaneously capture changes in reproductive output, developmental time and survival. We found significant decreases in population sizes for hosts exposed to live versus heat-killed S. aureus and found increased reproductive output after live P. aeruginosa exposure, compared with the corresponding heat-killed challenge. Nematode isolates with relatively higher population sizes after live P. aeruginosa infection produced fewer offspring after live S. aureus challenge. These findings reveal that wild C. elegans genotypes display a trade-off in defences against two distinct pathogen species that are evident in subsequent generations.

Dimethylsulfoniopropionate (DMSP) is an abundant organic sulfur metabolite produced by many phytoplankton species and degraded by bacteria via two distinct pathways with climate-relevant implications. We assessed the diversity and abundance of bacteria possessing these pathways in the context of phytoplankton community composition over a 3-week time period spanning September-October, 2014 in Monterey Bay, CA. The dmdA gene from the DMSP demethylation pathway dominated the DMSP gene pool and was harboured mostly by members of the alphaproteobacterial SAR11 clade and secondarily by the Roseobacter group, particularly during the second half of the study. Novel members of the DMSP-degrading community emerged from dmdA sequences recovered from metagenome assemblies and single-cell sequencing, including largely uncharacterized gammaproteobacteria and alphaproteobacteria taxa. In the DMSP cleavage pathway, the SAR11 gene dddK was the most abundant early in the study, but was supplanted by dddP over time. SAR11 members, especially those harbouring genes for both DMSP degradation pathways, had a strong positive relationship with the abundance of dinoflagellates, and DMSP-degrading gammaproteobacteria co-occurred with haptophytes. This in situ study of the drivers of DMSP fate in a coastal ecosystem demonstrates for the first time correlations between specific groups of bacterial DMSP degraders and phytoplankton taxa.

The co-evolution of bacterial plant pathogens and their hosts is a complex and dynamic process. Host resistance imposes stress on invading pathogens that can lead to changes in the bacterial genome enabling the pathogen to escape host resistance. We have observed this phenomenon with the plant pathogen Pseudomonas syringae pv. phaseolicola where isolates that have lost the genomic island PPHGI-1 carrying the effector gene avrPphB from its chromosome are infective against previously resistant plant hosts. However, we have never observed island extinction from the pathogen population within a host suggesting the island is maintained. Here, we present a mathematical model which predicts different possible fates for the island in the population; one outcome indicated that PPHGI-1 would be maintained at low frequency in the population long term, if it confers a fitness benefit. We empirically tested this prediction and determined that PPHGI-1 frequency in the bacterial population drops to a low but consistently detectable level during host resistance. Once PPHGI-1-carrying cells encounter a susceptible host, they rapidly increase in the population in a negative frequency-dependent manner. Importantly, our data show that mobile genetic elements can persist within the bacterial population and increase in frequency under favourable conditions.

The apoplast is the arena in which endophytic pathogens such as Pseudomonas syringae grow and interact with plant cells. Using metabolomic and ion analysis techniques, this study shows how the composition of Phaseolus vulgaris leaf apoplastic fluid changes during the first six hours of compatible and incompatible interactions with two strains of P. syringae pv. phaseolicola (Pph) that differ in the presence of the genomic island PPHGI-1. Leaf inoculation with the avirulent island-carrying strain Pph 1302A elicited effector-triggered immunity (ETI) and resulted in specific changes in apoplast composition, including increases in conductivity, pH, citrate, γ-aminobutyrate (GABA) and K(+) , that are linked to the onset of plant defence responses. Other apoplastic changes, including increases in Ca(2+) , Fe(2/3+) Mg(2+) , sucrose, β-cyanoalanine and several amino acids, occurred to a relatively similar extent in interactions with both Pph 1302A and the virulent, island-less strain Pph RJ3. Metabolic footprinting experiments established that Pph preferentially metabolizes malate, glucose and glutamate, but excludes certain other abundant apoplastic metabolites, including citrate and GABA, until preferred metabolites are depleted. These results demonstrate that Pph is well-adapted to the leaf apoplast metabolic environment and that loss of PPHGI-1 enables Pph to avoid changes in apoplast composition linked to plant defences.

The Columbia River has a dynamic and fast flushing estuary impacted by strong advection and mixing of riverine and oceanic waters, and high but variable loads of suspended particulate matter. Transient, but recurring water and nutrient fluxes from end-members impart strong spatial and temporal gradients, contributing to microbiological hotspots that play important ecological roles in the estuary. Investigations of corresponding microbiota require precisely timed samples that are contextualized by physical and biogeochemical data. To accomplish this, we embedded a robotic microbial sampler (Environmental Sample Processor, ESP) within the operations of an interdisciplinary observation and prediction system (Science and Technology University Research Network, SATURN; www.stccmop.org/saturn). Autonomous, adaptively sampled water collection by the ESP was implemented based on environmental conditions assessed from SATURN physical and biogeochemical sensors. Water was pumped from multiple depths to sensors and the ESP on dry land. If water met user-defined parameters, ESP sampling was automatically initiated. This strategy was tested during three deployments in summer 2013, during which operational tools for analysis and visualization were used to formulate well-constrained mission plans by providing estimates of the intensity and timing of oxygen-depleted ocean water intrusion and estuarine turbidity maxima. This allowed the effective characterization of the impact of these events on selected estuarine microbiota.

Wildfires release substantial quantities of carbon (C) into the atmosphere but they also convert part of the burnt biomass into pyrogenic organic matter (PyOM). This is richer in C and, overall, more resistant to environmental degradation than the original biomass, and, therefore, PyOM production is an efficient mechanism for C sequestration. The magnitude of this C sink, however, remains poorly quantified, and current production estimates, which suggest that ~1-5% of the C affected by fire is converted to PyOM, are based on incomplete inventories. Here, we quantify, for the first time, the complete range of PyOM components found in-situ immediately after a typical boreal forest fire. We utilized an experimental high-intensity crown fire in a jack pine forest (Pinus banksiana) and carried out a detailed pre- and postfire inventory and quantification of all fuel components, and the PyOM (i.e., all visually charred, blackened materials) produced in each of them. Our results show that, overall, 27.6% of the C affected by fire was retained in PyOM (4.8 ± 0.8 t C ha(-1)), rather than emitted to the atmosphere (12.6 ± 4.5 t C ha(-1)). The conversion rates varied substantially between fuel components. For down wood and bark, over half of the C affected was converted to PyOM, whereas for forest floor it was only one quarter, and less than a tenth for needles. If the overall conversion rate found here were applicable to boreal wildfire in general, it would translate into a PyOM production of ~100 Tg C yr(-1) by wildfire in the global boreal regions, more than five times the amount estimated previously. Our findings suggest that PyOM production from boreal wildfires, and potentially also from other fire-prone ecosystems, may have been underestimated and that its quantitative importance as a C sink warrants its inclusion in the global C budget estimates.

Metal hyperaccumulation is an unusual trait involving the uptake and storage of high concentrations of metals in the aerial tissues of plants. A number of hypotheses have been proposed to explain the evolution of the metal hyperaccumulation trait, of which the hypothesis that accumulated metal provides a defence against herbivores or pathogens has received most attention and support. Metal hyperaccumulation requires a range of physiological adaptations that enable plants to take up, transport, sequester and tolerate high concentrations of metal. Such adaptations may confer a fitness cost, and it has been suggested that metal hyperaccumulator plants may compensate for this cost by reducing investment in other traits such as induced disease resistance. This is supported by recent work that shows that metal hyperaccumulators such as Noccaea spp. show reduced or altered production of key components of induced disease resistance. However, an alternative explanation exists, which is that the physiological adaptations involved in metal hyperaccumulation require alterations to, or compromise, functional aspects of plant defence mechanisms. Here, the evidence for trade-offs between metal hyperaccumulation and disease resistance mechanisms is reviewed, and the nature of the physiological adaptations involved in metal hyperaccumulation and their potential to impact other forms of plant defence is discussed. It is suggested that defensive trade-offs may have been key to the evolution of the metal hyperaccumulation trait, resulting in increased dependence upon the protection conferred by metals.

CYP238A1, one of the two P450 enzymes in the genome of Pseudomonas putida KT2440, has been produced heterologously in Escherichia coli, purified, and found to bind acyclic and cyclic terpene alcohols such as farnesol, nerolidol, linalool, and terpineol. The other P450 enzyme in this organism (gene locus: PP1950) was also produced in E. coli but no substrate has been identified from a limited screen. A phthalate family oxygenase reductase (PFOR) encoded by the PP1957 gene, just downstream of the PP1955 gene for CYP238A1, accepts electrons from the reduced form of both nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate and is able to support monooxygenase activity of CYP238A1, both in vitro and in E. coli, in which both enzymes are produced. CYP238A1 oxidizes cis- and trans-nerolidol to the 9-hydroxy product, with no evidence of attack at the olefinic double bonds. The NADH turnover rate of 170 nmol(nmol-P450)⁻¹ Min⁻¹ for CYP238A1 with cis-nerolidol as substrate at a PP1957:CYP238A1 concentration ratio of 8:1 suggests that this PFOR could function as the physiological redox partner for CYP238A1. The physiological role of CYP238A1 may be related to the PP1955 gene being part of an island/cluster of inducible genes associated with energy metabolism and response to xenobiotics.

Antagonistic co-evolution between hosts and parasites (reciprocal selection for resistance and infectivity) is hypothesized to play an important role in host range expansion by selecting for novel infectivity alleles, but tests are lacking. Here, we determine whether experimental co-evolution between a bacterium (Pseudomonas fluorescens SBW25) and a phage (SBW25Φ2) affects interstrain host range: the ability to infect different strains of P. fluorescens other than SBW25. We identified and tested a genetically and phenotypically diverse suite of co-evolved phage variants of SBW25Φ2 against both sympatric and allopatric co-evolving hosts (P. fluorescens SBW25) and a large set of other P. fluorescens strains. Although all co-evolved phage had a greater host range than the ancestral phage and could differentially infect co-evolved variants of P. fluorescens SBW25, none could infect any of the alternative P. fluorescens strains. Thus, parasite generalism at one genetic scale does not appear to affect generalism at other scales, suggesting fundamental genetic constraints on parasite adaptation for this virus. © 2012 The Authors. Journal of Evolutionary Biology © 2012 European Society For Evolutionary Biology.

By inhibiting soil enzymes, tannins play an important role in soil carbon (C) and nitrogen (N) mineralization. The role of tannin chemistry in this inhibitory process, in conjunction with enzyme classes and isoforms, is less well understood. Here, we compared the inhibition efficiencies of mixed tannins (MTs, mostly limited to angiosperms) and condensed tannins (CTs, produced mostly by gymnosperms) against the potential activity of β-glucosidase (BG), N-acetyl-glucosaminidase (NAG), and peroxidase in two soils that differed in their vegetation histories. Compared with CTs, MTs exhibited 50% more inhibition of almond (Prunus dulcis) BG activity and greater inhibition of the potential NAG activity in the gymnosperm-acclimatized soils. CTs exhibited lower BG inhibition in the angiosperm-acclimated soils, whereas both types of tannins exhibited higher peroxidase inhibition in the angiosperm soils than in gymnosperm soils. At all of the tested tannin concentrations, irrespective of the tannin type and site history, the potential peroxidase activity was inhibited two-fold more than the hydrolase activity and was positively associated with the redox-buffering efficiency of tannins. Our finding that the inhibitory activities and mechanisms of MTs and CTs are dependent on the vegetative history and enzyme class is novel and furthers our understanding of the role of tannins and soil isoenzymes in decomposition.

Pseudomonas syringae pv. tomato DC3000 is a bacterial pathogen of Arabidopsis and tomato that grows in the apoplast. The non-protein amino acid γ-amino butyric acid (GABA) is produced by Arabidopsis and tomato and is the most abundant amino acid in the apoplastic fluid of tomato. The DC3000 genome harbors three genes annotated as gabT GABA transaminases. A DC3000 mutant lacking all three gabT genes was constructed and found to be unable to utilize GABA as a sole carbon and nitrogen source. In complete minimal media supplemented with GABA, the mutant grew less well than wild-type DC3000 and showed strongly reduced expression of hrpL and avrPto, which encode an alternative sigma factor and effector, respectively, associated with the type III secretion system. The growth of the gabT triple mutant was weakly reduced in Arabidopsis ecotype Landberg erecta (Ler) and strongly reduced in the Ler pop2-1 GABA transaminase-deficient mutant that accumulates higher levels of GABA. Much of the ability to grow on GABA-amended minimal media or in Arabidopsis pop2-1 leaves could be restored to the gabT triple mutant by expression in trans of just gabT2. The ability of DC3000 to elicit the hypersensitive response (HR) in tobacco leaves is dependent upon deployment of the type III secretion system, and the gabT triple mutant was less able than wild-type DC3000 to elicit this HR when bacteria were infiltrated along with GABA at levels of 1 mm or more. GABA may have multiple effects on P. syringae-plant interactions, with elevated levels increasing disease resistance.

The Mycorrhiza Helper Bacterium (MHB) Pseudomonas fluorescens BBc6R8 promotes the ectomycorrhizal symbiosis between Douglas fir roots and Laccaria bicolor. In this study, we identified a non-flagellar type III secretion system (T3SS) in the draft genome of BBc6R8 similar to that described in the biocontrol strain P. fluorescens SBW25. We examined whether this T3SS plays a role in the BBc6R8 mycorrhizal helper effect by creating a deletion in the rscRST genes encoding the central channel of the injectisome. The in vitro effect of BBc6R8 T3SS mutants on the radial growth rate of L. bicolor was unchanged compared with the parental strain. In contrast, T3SS mutants were unable to promote mycorrhization, suggesting that type III secretion plays an important role in the mycorrhizal helper effect of P. fluorescens BBc6R8 independent of the promotion of hyphal growth that BBc6R8 exhibits in vitro.

Microorganisms are key players in many biogeochemical processes and they strongly affect the health of soils and plants in many ways. Microbes are responsible for many soil activities, including decomposing dead plant tissues as saprophytes, but they can also attack living plants and cause plant diseases with great economic losses. On the other hand, some microbes can also protect crop plants against their pathogens and can improve the nutritional status of the plant (Bloemberg and Lugtenberg, 2001). This special issue of Microbial Biotechnology addresses the diverse ways in which knowledge of plant‐associated microorganisms can be applied to benefit agriculture, industry and the environment. Manipulation of microbial properties and modifications in microbial populations in the plant rhizosphere may lead to new interesting and exciting approaches for disease control and plant growth promotion. Biochemical characterization of the molecular mechanisms involved in plant–microbe interactions is uncovering chemicals and enzymes with a wide range of industrial applications. Finally, plant‐associated microorganisms can have ecosystem‐level effects on plants and soil, which can be exploited to improve soil quality, enhance carbon sequestration and to achieve bioremediation of pollutants, such as hydrocarbons and toxic metals. Many of the fields, in which knowledge about plant–microbe interactions have been applied, are reviewed in this issue by Wu and co‐workers (2009), including applications in biofertilization, plant disease control, production of bioactive compounds, remediation and carbon sequestration.

Nitrilase enzymes (nitrilases) catalyse the hydrolysis of nitrile compounds to the corresponding carboxylic acid and ammonia, and have a wide range of industrial and biotechnological applications, including the synthesis of industrially important carboxylic acids and bioremediation of cyanide and toxic nitriles. Nitrilases are produced by a wide range of organisms, including plants, bacteria and fungi, but despite their biotechnological importance, the role of these enzymes in living organisms is relatively underexplored. Current research suggests that nitrilases play important roles in a range of biological processes. In the context of plant-microbe interactions they may have roles in hormone synthesis, nutrient assimilation and detoxification of exogenous and endogenous nitriles. Nitrilases are produced by both plant pathogenic and plant growth-promoting microorganisms, and their activities may have a significant impact on the outcome of plant-microbe interactions. In this paper we review current knowledge of the role of nitriles and nitrilases in plants and plant-associated microorganisms, and discuss how greater understanding of the natural functions of nitrilases could be applied to benefit both industry and agriculture.

Rehabilitation and reforestation of disused forest roads and landings can be facilitated by the incorporation of organic matter. The British Columbia forest industry creates residual woody materials, but they are nutrient poor and may leach phenolic compounds. We assessed the potential for wood wastes (chipped cedar wood waste, sort-yard waste, hogfuel) and co-composts with shellfish waste or municipal biosolids to provide inorganic N and release phenolics and condensed tannins, compared with natural forest floor and mineral soil. Initial concentrations of tannins and phenolics were low, and 13C cross-polarization and magic-angle spinning nuclear magnetic resonance spectroscopy showed that composts were still dominated by wood. During a 426-d laboratory leaching experiment, release of phenolics from woody amendments (other than cedar wood) was lower than from native forest floor. The pH levels of woody amendments and their leachates were also within the range of native forest floor and soil (except cedar wood, which was the most acidic material). Co-composts had higher total N and available P, greatly reduced tannins and phenolics, and negligible leaching of polyphenols. Uncomposted materials released very little N during the incubation. Hogfuel-biosolids compost released a large amount of nitrate, but only during the first 100 d. Shrimp-wood compost released moderate amounts of ammonium and nitrate throughout the incubation, had high available P and low tannin content, and released less polyphenols than did native forest floors. Our results indicate that appropriate use of these amendments does not pose an environmental risk with regard to the parameters measured in this study.

Summary • The role of litter tannins in controlling soil nitrogen (N) cycling may explain the competitive ability of Kalmia relative to black spruce (Picea mariana), although this has not been demonstrated experimentally. • Here, the protein-precipitation capacities of purified tannins and leaf extracts from Kalmia and black spruce were compared. The resistance to degradation of tannin–protein precipitates from both species were compared by monitoring carbon (C) and N dynamics in humus amended with protein, purified tannins or protein–tannin precipitates. The purity of the precipitates was verified using solid-state 13C nuclear magnetic resonance (NMR) spectra. The ability of mycorrhizal fungi associated with both species to grow on media amended with tannin–protein complexes as the principal N source was also compared. • The protein precipitation capacity of Kalmia tannins was superior to those of black spruce. Humus amended with protein increased both mineral and microbial N, whereas humus amended with tannin–protein precipitates increased dissolved organic N. Mycorrhizal fungi associated with Kalmia showed better growth than those associated with black spruce when N was provided as tannin–protein precipitates. • These data suggest that Kalmia litter increases the amount of soil N sequestered as tannin–protein complexes, which may improve the competitive ability of Kalmia relative to black spruce by favouring N uptake by mycorrhizas associated with the former.

During an initial encounter with herpes simplex virus type 1 (HSV-1) it takes several days for an adaptive immune response to develop and for herpes-specific CD8(+) T cells to infiltrate sites of infection. By this time the virus has firmly established itself within the innervating sensory nervous system where it then persists indefinitely. Preventing the establishment of viral latency would require blocking the skin to nervous system transmission of the virus. We wished to examine if CD8(+) T cells present early during acute HSV-1 infection could block this transmission. We show that effector CD8(+) T cells failed to prevent the establishment of HSV latency even when present prior to infection. This lack of blocking likely reflects the delayed infiltration of the CD8(+) T cells into the infected skin. Examination of the kinetics of HSV-1 infection highlighted the rapidity at which the virus infects the sensory ganglia and singled out early viral replication within the skin as an important factor in determining the magnitude of the ensuing latent infection. Though unable to prevent the establishment of latency, CD8(+) T cells could reduce the average viral copy number of the residual latent infection by dampening the skin infection and thus limiting the skin-to-nerve transmission of virus.

In vivo expression technology (IVET) analysis of rhizosphere-induced genes in the plant growth-promoting rhizobacterium (PGPR) Pseudomonas fluorescens SBW25 identified a homologue of the type III secretion system (TTSS) gene hrcC. The hrcC homologue resides within a 20-kb gene cluster that resembles the type III (Hrp) gene cluster of Pseudomonas syringae. The type III (Rsp) gene cluster in P. fluorescens SBW25 is flanked by a homologue of the P. syringae TTSS-secreted protein AvrE. P. fluorescens SBW25 is non-pathogenic and does not elicit the hypersensitive response (HR) in any host plant tested. However, strains constitutively expressing the rsp-specific sigma factor RspL elicit an AvrB-dependent HR in Arabidopsis thaliana ecotype Col-0, and a host-specific HR in Nicotiana clevelandii. The inability of wild-type P. fluorescens SBW25 to elicit a visible HR is therefore partly attributable to low expression of rsp genes in the leaf apoplast. DNA hybridization analysis indicates that rsp genes are present in many plant-colonizing Pseudomonas and PGPR, suggesting that TTSSs may have a significant role in the biology of PGPR. However, rsp and rsc mutants retain the ability to reach high population levels in the rhizosphere. While functionality of the TTSS has been demonstrated, the ecological significance of the rhizosphere-expressed TTSS of P. fluorescens SBW25 remains unclear.

Summary The recent isolation of the ammonia-oxidizing crenar- chaeon Nitrosopumilus maritimus has expanded the known phylogenetic distribution of nitrifying pheno- types beyond the domain Bacteria. To further charac- terize nitrification in the marine environment and explore the potential crenarchaeal contribution to this process, we quantified putative nitrifying genes and phylotypes in picoplankton genomic libraries and environmental DNA samples from coastal and open ocean habitats. Betaproteobacteria ammonia monooxygenase subunit A (amoA) gene copy numbers were low or undetectable, in stark contrast to crenarchaeal amoA-like genes that were broadly dis- tributed and reached up to 6 ¥ 10 4 copies ml -1 . Unex- pectedly, in the North Pacific Subtropical Gyre, a deeply branching crenarchaeal group related to a hot spring clade (pSL12) was at times abundant below the euphotic zone. Quantitative data suggested that the pSL12 relatives also contain archaeal amoA-like genes. In both coastal and open ocean habitats, close relatives of known nitrite-oxidizing Nitrospina species were well represented in genomic DNA libraries and quantitative PCR profiles. PlanktonicNitrospina depth distributions correlated with those of Crenarchaea. Overall, the data suggest that amoA-containing Cre- narchaea are more phylogenetically diverse than pre- viously reported. Additionally, distributional patterns of planktonic Crenarchaea and Nitrospina species suggest potential metabolic interactions between these groups in the ocean's water column.

Limited studies have been carried out on the level of vitamin D in non-human primates. Herein, we present data using the preferred vitamin D indicator, serum 25-OH vitamin D, sequentially collected...

ing the sustainable use of rangelands for livestock production. However, many ecosystem components and Growing interest in the potential for soils to provide a sink for processes like plant community structure, soil properatmospheric C has prompted studies of effects of management on the amountandnatureofsoilorganicC(SOC).Inthisstudy,weevaluated ties, and nutrient cycling are also affected by grazing effects of different grazing management regimes (light grazing [LG], management (Schuman et al., 1999). Density and duraheavy grazing [HG], and non-grazed exclosures [EX]) on amount tion of rangeland stocking can affect plant community and composition of SOC at the USDA–ARS High Plains Grasslands compositionthroughdisplacementofcool-seasonmixed ResearchStation (HPGRS),Cheyenne, WY.Soils (0–5cm) fromeach grassesbywarm-seasonshortgrassesinnorthernmixedtreatment were analyzed for total C and N contents and lignin com- grass prairies (Dormaar and Willms, 1990). position. Soil organic C and N contents were significantly greater in Grazing can also influence the amount and composiLG (SOC–13.8 Mg ha 1 ; total N–1.22 Mg ha 1

Fertilization elicits a dramatic, transient rise in Ca2+ within the egg which is an essential component of egg activation and consequent initiation of development. In the sea urchin egg, three distinct Ca2+ stores have been identified which could, either individually or in combination, initiate Ca2+ release at fertilization. Inositol 1,4,5-trisphosphate (IP3) production by phospholipase C (PLC) has been suggested as the singular signal in initiating the Ca2+ transient. Other studies indicate that Ca2+ stores gated by cyclic adenosine diphosphate ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP) are also necessary. We have examined the temporal relationship between the Ca2+ rise and IP3 production at fertilization in vivo within individual eggs using a green fluorescent protein (GFP) coupled to a pleckstrin homology (PH) domain that can detect changes in IP3. Translocation of the probe occurred after the Ca2+ rise was initiated. Earlier, and possibly smaller, IP3 changes could not be excluded due to limitations in probe sensitivity. High IP3 levels are maintained during the decline in cytoplasmic Ca2+, suggesting that later IP3 metabolism might not be related to regulation of Ca2+, but may function to modulate other PIP2 regulated events such as actin polymerization or reflect other novel phosphoinositide signaling pathways.

Phasing out beehive burners and rising costs for landfilling have increased the need to widen options for utilization of the smaller size fractions of woody wastes generated during log handling and sawmilling in British Columbia. We characterized several size classes of logyard fines up to 16 mm sampled from coastal and interior operations. Total C, total N, ash, and condensed tannin concentrations were consistent with properties derived largely from wood, with varying proportions of bark and mixing with mineral soil. Especially for < 3-mm fractions, the latter resulted in high ash contents that would make them unsuitable for fuel. Solid-state 13C cross-polarization magic-angle spinning (CPMAS) nuclear magnetic resonance (NMR) spectra were consistent with the chemical data, with high O-alkyl intensity and similarity to naturally occurring woody forest floor; no samples were high in aromatic or phenolic C. Aqueous extracts of two < 16-mm fines, which accounted for only a small proportion of the total C, were enriched in alkyl C and had low or undetectable tannins. Application to forest sites might cause short-term immobilization of N, but also might include possible longer-term benefits from reduction of N loss after harvesting and restoration of soil organic matter in degraded sites.

Polyribonucleotide probes targeting planktonic archaeal (Group I and II) and bacterial rRNA revealed that Ar- chaea comprised a significant fraction of total prokaryote cell abundance in the marine waters west of the Antarctic Peninsula. Determinations of Archaea and Bacteria cell abundances were made during two research cruises to the Palmer Long-Term Ecological Research region during the austral winter and summer of 1999. During the austral summer, surface water abundances of Group I (GI) Archaea were generally low, averaging 4.7 3 10 3 cells ml 21 and comprising 9-39% of the total picoplankton abundance in the meso- (150-1,000 m) and bathypelagic (1,000-3,500 m) circumpolar deep water (CDW). Relative to summertime distri- butions, GI cells were more evenly distributed throughout the water column during the winter, averaging 10% of the picoplankton in the surface waters and 13% in the CDW. Surface water GI abundance increased 44% between the summer and winter, coincident with a fivefold decrease in GI abundance in the deeper waters. The abundance of Group II (GII) Archaea was persistently ,2% of the total picoplankton throughout the water column in both summer and winter. Bacterial abundance was greatest in the upper water column (0-100 m) during the summer, averaging 3.9 3 10 5 cells ml 21 and comprised 89% of the total picoplankton assemblage. Generally, GI Archaea varied seasonally in the deeper waters, whereas bacterial abundance varied more in the upper waters. The observed variability in bacterial and archaeal abundance suggests that these two groups of marine picoplankton are dynamic components of Southern Ocean microbial food webs.

The protozoon Naegleria gruberi is able to carry out amoeboid locomotion at the water-air interface in a manner indistinguishable from that exhibited on solid substrata with the production of focal contacts and associated filopodia. The speed of locomotion at this interface can be modulated by changes in electrolyte concentrations; these speed changes are identical to those observed at a water-glass interface. The nature of the water-air interface is discussed leading to the hypothesis that surface tension alone could provide suitable properties for the adhesion and translocation of amoebae at this interface without necessitating specific, absorbed molecules. The temporary swimming flagellate stage of Naegleria is able to dock at the interface, make stable adhesions to it, and revert to the amoeboid phenotype. Conversely, amoebae resident at the water-air interface can transform to swimming flagellates and escape into the bulk liquid phase. We report the presence of Naegleria amoebae in the surface microlayers of natural ponds; thus, in freshwater bodies there may be active shuttling of Naegleria amoebae from the benthos to the surface microlayers by means of the non-feeding, swimming flagellate phenotype. The public health implication of this behaviour in the case of the pathogenic relative, Naegleria fowleri, is discussed.

Photosynthetic microorganisms play a crucial role in the marine environment. In vast areas of the oceans, marine primary productivity is performed by cells smaller than 2-3 micro m (picoplankton). Here, we report on molecular analyses of the conserved photosynthetic psbA gene (coding for protein D1 of photosystem II reaction centre) as a diversity indicator of naturally occurring marine oxygenic picophytoplankton. The psbA genes proved to be good indicators of the presence of a wide variety of photosynthetic marine microbial groups, including new cyanobacterial groups and eukaryotic algae (prasinophytes). Furthermore, using environmental bacterial artificial chromosome (BAC) libraries, we were able to correlate psbA genes with small subunit rRNAs and, therefore, to confirm their phylogenetic affiliation.

As part of investigations into the effects of harvesting old-growth forest, we characterized carbon in five organic matter pools in eight forest chronosequences of coastal British Columbia. Each chronosequence comprised stands in four seral stages from regeneration (3-8 yr) to old-growth (>250 yr), with second-growth stands mostly of harvest origin. Stands were located in two biogeoclimatic subzones with contrasting climate (wetter, slightly cooler conditions on the west coast of Vancouver Island than on the east). Carbon concentrations in fine woody debris (FWD), forest floor (LFH), fine roots from LFH, and two water-floatable fractions from 10 to 30 cm mineral soil (MIN-ROOT, 2-8 mm and MIN-FLOAT

Recommended dietary intake of most nutrients is satisfied by a majority of Puerto Rican schoolchildren, however this is not true for all nutrients. In this presentation we examine the contribution ...

Biosolids are effective forest fertilizers. In order to facilitate their use it is important that one be able to predict the amount and rate of mineralization of nutrients, particularly nitrogen, and the relationship between substrate chemistry and N release. We examined the relationships between substrate quality and nitrogen release in a variety of organic materials. Rates of decomposition and net N mineralization from four biosolids, wheat straw, paper fines, and Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] needle litter were measured during 391-d incubations in a greenhouse, and at two field sites in wet coastal and dry interior forests. Decomposition rates were best predicted by a model incorporating the ratio of carbon to organic matter. The decomposition model extrapolated well to the field when site-specific correction factors were applied. There was a weak relationship between rates of decomposition and net N mineralization. Rates of net N mineralization were best predicted by a model incorporating the initial organic N concentration and the proportion of phenolic C determined from solid-state 13C nuclear magnetic resonance (NMR) spectroscopy. The mineralization model extrapolated less well to the field, but the effect of substrate chemistry was still apparent. Among the four biosolids there was a strong correlation between organic N concentration and indices or protein determined from 13C NMR, suggesting that these protein indices may be useful for predicting N mineralization from biosolids. There was some evidence that the protein content and N mineralization in biosolids may be predictable from the sewage treatment process employed.

Pseudomonas syringae pv. tomato and the closely related pathovar P. s. pv. maculicola have been the focus of intensive research in recent years, not only because of the diseases they cause on tomato and crucifers, but because strains such as P. s. pv. tomato DC3000 and P. s. pv. maculicola ES4326 are pathogens of the model plant Arabidopsis thaliana. Consequently, both P. s. pv. tomato and P. s. pv. maculicola have been widely used to study the molecular mechanisms of host responses to infection. Analyses of the molecular basis of pathogenesis in P. s. pv. tomato reveal a complex and intimate interaction between bacteria and plant cells that depends on the coordinated expression of multiple pathogenicity and virulence factors. These include toxins, extracellular proteins and polysaccharides, and the translocation of proteins into plant cells by the type III (Hrp) secretion system. The contribution of individual virulence factors to parasitism and disease development varies significantly between strains. Application of functional genomics and cell biology to both pathogen and host within the P. s. pv. tomato/A. thaliana pathosystem provides a unique opportunity to unravel the molecular interactions underlying plant pathogenesis. Taxonomic relationship: Bacteria; Proteobacteria; gamma subdivision; Pseudomonadaceae/Moraxellaceae group; Pseudomonadaceae family; Pseudomonas genus; Pseudomonas syringae species; tomato pathovar. Microbiological properties: Gram-negative, aerobic, motile, rod-shaped, polar flagella, oxidase negative, arginine dihydrolase negative, DNA 58–60 mol% GC, elicits the hypersensitive response on tobacco. Host range: Primarily studied as the causal agent of bacterial speck of tomato and as a model pathogen of A. thaliana, although it has been isolated from a wide range of crop and weed species. Disease symptoms: Tomato (Lycopersicon esculentum): Brown-black leaf spots sometimes surrounded by chlorotic margin; dark superficial specks on green fruit; specks on ripe fruit may become sunken, and are surrounded by a zone of delayed ripening. Stunting and yield loss, particularly if young plants are infected. Reduced market value of speckled fruit. A. thaliana: Water-soaked, spreading lesions, sometimes surrounded by chlorotic margin. Epidemiology: Seed borne. Survives as a saprophyte in plant debris, soil and on leaf surfaces. Dispersed by aerosols and rain splash. Development of disease symptoms favoured by leaf wetness and cool temperatures (55–77 °F/13–25 °C). Disease control: Pathogen-free seed and transplants. Resistant and tolerant cultivars. Sanitation, rotation, and drip irrigation to reduce leaf wetness. Some measure of control with bactericides (copper, streptomycin).

Degradiation of [U-13C] glucose in sphagnum majus litter : responses to redox, pH and temperature.

Summary As a source of organic matter, crop residues affect the behaviour of pesticides in agricultural soils. The fate of [U-ring-13C] and [U-ring-14C] atrazine (6-chloro-N-ethyl-N-isopropyl-1,3,5-triazine-2,4-diamine) was investigated during laboratory incubation under controlled conditions in a loamy soil amended with wheat straw at two different states of decomposition: no preliminary decomposition or 6 months’ preliminary decomposition. After 3 months, non-extractable, so-called ‘bound’, 13C-atrazine residues were recovered in three particle-size fractions (> 200, 50–200 and 200 and 50–200 μm contained significant amounts of bound residues, especially when straw was added to the soil. CP/MAS 13C-NMR analysis of humic acids obtained from

Attenuated vaccines are an important means of controlling Theileria annulata infection of cattle. Production is by prolonged cultivation of macroschizont-infected cells. The mechanism of attenuation remains unclear. There are three general nonmutually exclusive possibilities: Selection of avirulent subpopulations, genome rearrangements and alterations in gene expression. Several groups, including ours, have provided evidence that the population structure usually tends to simplify during attenuation. Our data on the T. annulata (Ta) Ankara cell line show that attenuation is not necessarily accompanied by the population becoming clonal. We have been unable to detect large DNA rearrangements. Evidence for alterations in host and parasite gene expression during attenuation is available. With respect to the host we have shown that attenuation is accompanied by loss of expression of parasite induced matrix metalloproteinases (MMPs). However, in different lines different protease activities are involved. In the T. annulata Ode line we have shown that 8 activities (including MMP9) are downregulated and that this correlates with a loss of metastatic behaviour. This has previously been shown in vitro using reconstituted basement membrane (Matrigel) and is demonstrated in vivo using scid mice in this study. Thus part of the pathology, namely the ability to disseminate, mediated by host MMPs, is lost upon attenuation. Re-isolation experiments have shown that the reduction/loss of MMP is a stable transferable trait. A logical extension is that loss of MMP activity (and virulence in general) must be at the most fundamental level a genetic trait of the parasite. Evidence for loss of parasite gene expression is implied by the loss of the ability to differentiate into merozoites on attenuation. Specific evidence for loss of parasite gene expression has been obtained using differential RNA display. We view virulence as a multifactorial phenomenon involving interacting subpopulations of cells and attenuation is a threshold effect whereby the number of virulence factors is reduced below a critical level. On this basis there will be many different ways to achieve attenuation.

We examined the impacts of possible future land development patterns on the biodiversity of a landscape. Our landscape data included a remote sensing derived map of the current habitat of the study area and six maps of future habitat distributions resulting from different land development scenarios. Our species data included lists of all bird, mammal, reptile, and amphibian species in the study area, their habitat associations, and area requirements for each. We estimated the area requirements using home ranges, sampled population densities, or genetic area requirements that incorporate dispersal distances. Our measures of biodiversity were species richness and habitat abundance. We calculated habitat abundance in two ways. First, we computed the total habitat area for each species in each landscape. Second, we calculated the number of habitat units for each species in each landscape by dividing the size of each habitat patch in the landscape by the area requirement and summing over all patches. Species richness was based on presence of habitat. Species became extinct in the landscape if they had no habitat area or no habitat units, respectively. We then computed ratios of habitat abundance in each future landscape to habitat abundance in the present for each species. We also computed the ratio of future to present species richness. We then calculated summary statistics across all species. Species richness changed little from present to future. There were distinctly greater risks to habitat abundance in landscapes that extrapolated from present trends or zoning patterns, however, as opposed to landscapes in which land development activities followed more constrained patterns. These results were stable when tested using Monte Carlo simulations and sensitivity tests on the area requirements. We conclude that this methodology can begin to discriminate the effects of potential changes in land development on vertebrate biodiversity.

Summary Nitric oxide (NO) was produced when bovine peripheral blood mononuclear cells (PBMC) or purified, adherent PBMC (macrophages) were incubated in vitro with bovine recombinant interferon gamma (Bo rIFN-γ). NO was produced by cells from naive, uninfected calves as well as by cells from cattle either infected with or recovered from infection with Theileria annulata or Theileria parva. PBMC of cattle undergoing tropical theileriosis (T. annulata infection) or East Coast fever (T. parva infection) synthesized NO spontaneously in vitro. NO was also induced when PBMC of immune, but not of naive, cattle were cultured with T. annulata macroschizont-infected cell lines. Macrophages alone were not stimulated to produce NO by such infected cells. In vitro establishment of macroschizont-infected cell lines was suppressed either by incubating sporozoites with S-nitroso-N-acetyl-DL-penicillamine (SNAP), a NO releasing molecule, prior to invasion of PBMC or by pulsing developing cultures of trophozoite-infected cells with SNAP. Proliferation of established macroschizont-infected cell lines was not affected by SNAP. Taken together with the well documented roles of NO in neurotransmission, vasodilatation, cell and tissue damage and immunosuppression, the results presented here indicate that NO may not only protect cattle against T. annulata and T. parva but, if produced in excess, play a prominent role in the pathogenesis of tropical theileriosis and East Coast fever.

Theorists have noted that cognitive processing and personality are essential in explaining television viewing behaviour, but evidence has been difficult to obtain. The present investigation employed a new cognitive-personality approach to the intriguing question why do we watch what we do? Because self is central to personality, concept of self was measured in addition to concepts of television genres. In two studies, university students rated self and various television genres on several bipolar constructs in a computerized grid test. They also indicated their viewing behaviour for each genre and their viewing hours per week. Indices of congruence between concept of self and concept of each genre (self—other distance scores) were calculated and correlated with genre viewing behaviour. The findings support the conclusion that similarity of constructions of self and genre predicts selective viewing and demonstrate the relevance of viewer's genre concepts and personality to television viewing behaviour.

Matrix-assisted laser desorption ionization of vitamin B12 at 337 nm yields low abundances of the protonated molecule in time-of-flight mass spectra when the matrix α-cyano-4-hydroxycinnamic acid is used. A variety of fragment ions are observed in the time-of-flight mass spectra which may be assigned on the basis of previous studies of vitamin B12 fragmentation. When the matrix 4-hydroxy-3-iodo-5-nitrobenzoic acid is used, an abundant signal from the protonated molecule ion of vitamin B12 is produced. Fragment ions are still observed in the time-of-flight mass spectra but at much lower abundances. Studies of the influence of matrix-to-analyte ratio and desorbing laser intensity argue against direct photodissociation as the mechanism of fragment ion formation. Matrix choice appears to most strongly influence the internal energies of the laser-desorbed ions thereby controlling the fragmentation processes.

Bovine macrophage-derived tumour necrosis factor-alpha/cachectin (TNF-alpha) was synthesized when peripheral blood mononuclear cells (PBMC) and purified adherent PBMC from naive and Theileria annulata-infected cattle were incubated in vitro with concanavalin A (Con-A) or bovine recombinant interferon gamma (Bo rIFN-gamma). TNF-alpha production was also induced when adherent PBMC were cultured with T. annulata macroschizont-infected cells. In contrast, non-adherent PBMC from sublethally infected cattle produced interferon (IFN) when incubated with Hu rIL-2, Con-A, phytohaemagglutinin (PHA) or T. annulata macroschizont-infected cells growing as cell lines in vitro. Whilst PBMC from lethally infected cattle spontaneously produced IFN-gamma during advanced stages of infection, the sera of such animals contained type 1 IFN (alpha/beta). IFN was also produced by T. annulata macroschizont-infected cell lines maintained in vitro. This work suggests that cytokines serve as crucial links between proliferating Theileira-infected cells and the characteristic clinical symptoms of tropical theileriosis.

We have observed that using nitrocellulose in sample preparation for matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry increases the yield of peptide [M + H]+ ions. Addition of nitrocellulose also provides improvement in sample-to-sample reproducibility of MALDI ion yield and improves the precision of peptide quantitation. Mass spectrometry and optical microscopy results suggest that the nitrocellulose modifies the crystallization of matrix/analyte solution to allow more even coverage over the sample surface.

The unit cell of N-tert-butyl-3-(tert-butylimino)-2-nitropropen-1-amine (4) is monoclinic, space group P2 1 /C, with a=9.669(4), b=16.415(6), c=17.341(7)A, and Z=4. Hydrolysis of 4 leads to (E)-3-(N-tert-butylamino)-2-nitro-2-propenal (5), whose unit cell is monoclinic, space group P2 1 /C, with a=6.821(1), b=20.707(6), c=6.303(1)A, and Z=4. Compounds 4 and 5 both possess C 2 symmetry in the solid state, their «U-shaped» conjugated cores being essentially planar. In each case this planarity is enforced by an intramolecular hydrogen bond