Application of subtraction hybridization for the development of a Rhizobium leguminosarum biovar phaseoli and Rhizobium tropici group-specific DNA probe

Abstract A combined subtraction hybridization and polymerase chain reaction/amplification technique was used to develop a DNA probe which was specific for the Rhizobium leguminosarum biovar phaseoli and the Rhizobium tropici group. Total genomic DNA preparations from Rhizobium leguminosarum biovar viciae, Rhizobium leguminosarum biovar trifolii, Rhizobium sp., Agrobacterium tumefaciens, Rhizobium fredii, Bradyrhizobium japonicum, Bradyrhizobium ssp. and Rhizobium meliloti were pooled and used as subtracter DNA against total genomic DNA from the Rhizobium leguminosarum biovar phaseolo strain KIM5s. Only one round of subtraction hybridization at 65°C was necessary to remove all cross-hybridizing sequences. Dot blot hybridizations with total genomic DNA of the eight subtracter organisms and 29 bacteria of different groups confirmed the high specificity of the isolated DNA sequences. Dot blot hybridizations and total genomic DNA from ten different R. Leguminosarum biovar phaseoli and R. tropici strains resulted in strong hybridization signals for all strains tested. The DNA probe for the R. tropici and R. leguminosarum biovar phaseoli group was used for dot blot hybridization with DNA extracts from three tropical and one boreal soil. When correlated with data from Most Probable Number analyses the probe was capable of detecting as low as 3 × 10⁴ homologous indigenous rhizobia per g soil. The technique offers great benefits for the development of DNA probes for monitoring bacterial populations in environmental samples.     

Ascaris suum: Nutrient absorption,growth, and intestinal pathology in young pigs experimentally infected with 15-day-old larvae

Three-week-old pigs on high (HP) or low (LP) protein diets were infected with 15-day-old Ascaris suum larvae (W). Including noninfected pigs (C), the experimental groups were HPW, LPW, HPC, and LPC. After 8 weeks, worm burden in the intestine averaged 42 in LPW and 31 in HPW. Nitrogen balance during Week 4 showed nonsignificantly less nitrogen absorption and retention in LPW compared to LPC. A similar, nonsignificant decrease in fat absorption was recorded in LPW vs LPC and in HPW vs HPC. The weight of the small intestine was significantly greater in W than C pigs but did not differ because of protein level. The weight correlated positively to worm burden and the increase was due mainly to hypertrophy of the tunica muscularis (muscle layers).     

Protein model discrimination attempts using mutational sensitivity,predicted secondary structure,and model quality information

Structure prediction methods often generate a large number of models for a target sequence. Even if the correct fold for the target sequence is sampled in this dataset, it is difficult to distinguish it from other decoy structures. An attempt to solve this problem using experimental mutational sensitivity data for the CcdB protein was described previously by exploiting the correlation of residue depth with mutational sensitivity (r ~ 0.6). We now show that such a correlation extends to four other proteins with localized active sites, and for which saturation mutagenesis datasets exist. We also examine whether incorporation of predicted secondary structure information and the DOPE model quality assessment score, in addition to mutational sensitivity, improves the accuracy of model discrimination using a decoy dataset of 163 targets from CASP. Although most CASP models would have been subjected to model quality assessment prior to submission, we find that the DOPE score makes a substantial contribution to the observed improvement. We therefore also applied the approach to CcdB and four other proteins for which reliable experimental mutational data exist and observe that inclusion of experimental mutational data results in a small qualitative improvement in model discrimination relative to that seen with just the DOPE score. This is largely because of our limited ability to quantitatively predict effects of point mutations on in vivo protein activity. Further improvements in the methodology are required to facilitate improved utilization of single mutant data.     

Nutrient loading of a forest soil

Summary During July 1988 rooted and non-rooted experimental chambers were established in a Norway spruce (Picea abies. Karst) stand in south Devon U.K. Replicates were supplemented with ammonium and nitrate. The leachates were analysed to monitor the release of mineral-N species and cations over the 17-week experimental period. Ammonium treatments leached 300% more calcium and magnesium than controls. The onset of nitrification resulted in a decrease in sodium losses from ammonium treatments reflecting a decrease in the exchanging capacity of the soil solution. These results are discussed in relation to mineral ion leaching in soils subjected to increesed N-loading, and the ability of soils to buffer these perturbations.     

Biochemical and genetic analysis of a Chlamydomonas reinhardtii mutant devoid of chloroplastic glutamine synthetase activity

A spontaneous double mutant of Chlamydomonas reinhardtii, designated ARF3, was resistant to L-methionine-S-sulfoximine (MSX), lacked chloroplastic glutamine synthetase (GS2) activity, and grew very poorly in all media tested. In segregants obtained after genetic crosses, the poor-growth phenotype was always linked to the lack of GS2 and to a diminished rate of consumption of ammonium, even under conditions where photorespiration was minimized. The ammonium permeases in mutant ARF3, however, were not altered. This indicates that, unlike in higher plants, GS2 contributes substantially to the primary assimilation of ammonia in this alga, and that its function cannot be replaced by the cytosolic glutamine synthetase. In genetic crosses, the MSX resistance and the lack of GS2 segregated independently, indicating that resistance was not due to an altered form of GS2. Received: 5 June 1998 / Accepted: 10 September 1998     

Effects of nitrogen supply and elevated carbon dioxide on construction cost in leaves of Pinus taeda (L.) seedlings

Seedlings of loblolly pine (Pinus taeda L.) were grown under varying conditions of soil nitrogen and atmospheric carbon dioxide availability to investigate the interactive effects of these resources on the energetic requirements for leaf growth. Increasing the ambient CO₂ partial pressure from 35 to 65 Pa increased seedling growth only when soil nitrogen was high. Biomass increased by 55% and photosynthesis increased by 13% after 100 days of CO₂ enrichment. Leaves from seedlings grown in high soil nitrogen were 7.0% more expensive on a g glucose g^–1 dry mass basis to produce than those grown in low nitrogen, while elevated CO₂ decreased leaf cost by 3.5%. Nitrogen and CO₂ availability had an interactive effect on leaf construction cost expressed on an area basis, reflecting source-sink interactions. When both resources were abundant, leaf construction cost on an area basis was relatively high (81.8±3.0 g glucose m^–2) compared to leaves from high nitrogen, low CO₂ seedlings (56.3±3.0 g glucose m^–2) and low nitrogen, low CO₂ seedlings (67.1±2.7 g glucose m^–2). Leaf construction cost appears to respond to alterations in the utilization of photoassimilates mediated by resource availability.     

Needle CO2 exchange, structure and defense traits in relation to needle age in Pinus heldreichii Christ – a relict of Tertiary flora

 Pinus heldreichii Christ is a long-lived, slow-growing Tertiary relict from the Balkans. In this study we evaluated the physiological characteristics of eight needle-age classes of P. heldreichii grown at the Arboretum of the Institute of Dendrology in Kórnik, Poland. At the end of the growing season, current-year foliage had the highest rates of mass-based light-saturated net photosynthesis (A_sat) of 33.5 nmol CO₂· g^–1· s^–1. A_sat decreased with needle age, but older needle classes retained from approximately 62 to 26% of the current needles’ rate. The relationship between leaf N and chlorophyll a concentration among all needle-age classes was highly significant (r = 0.96, P = 0.0006). The variation in A_sat of 1- to 7-year-old needles was linearly related to needle N concentration (r = 0.98, P = 0.0001). Needle dark respiration rates among these needle age classes ranged from 0.8 to 2.2 nmol · g^–1· s^–1 and decreased with needle age and nitrogen concentration. Total phenols and glucose concentrations increased linearly with needle age. A similar pattern was observed in acid buffering capacity and the pH of tissue homogenates. The water content ranged from 62% for the current needles to 51% for the 6-year-old needles. Greater investment in leaf structural tissue and increased chemical defense is associated with higher structural cost of older needles and may reduce their photosynthetic activity. Significant declines in water and nitrogen content with needle age and an increase in content of phenolics is most likely a defense adaptation of P. heldreichii related to the species’ long-lived leaves. Received: 8 January 1997 / Accepted: 4 March 1997     

Methodological approach for the assessment of environmental effects of agroforestry at the landscape scale

Silvoarable agroforestry, the deliberate combined use of trees and arable crops on the same area of land, has been proposed in order to improve the environmental performance of agricultural systems in Europe. Based on existing models and algorithms, we developed a method to predict the environmental effects of SAF at a farm and landscape scale. The method is comprised of an assessment of soil erosion, nitrogen leaching, carbon sequestration, and landscape diversity and allowed the comparison of the environmental performance of SAF with arable systems using these four indicators.The method was applied to three landscape test sites of 4 km × 4 km each in Spain, France, and The Netherlands, and compared different levels of agroforestry adoption on farmland of different potential productivity. Silvoarable agroforestry was predicted to reduce soil erosion by up to 70%, to reduce N leaching by 20–30%, to increase C sequestration over 60 years by up to 140 tonnes C ha⁻¹, and to increase landscape diversity up to four times. The method developed was executed with widely available landscape and farm structural data and can therefore be applied to other regions in order to obtain a broader assessment of the environmental performance of silvoarable agroforestry systems.     

Short-term accumulation of organic matter and nutrient contents in two dry sand ecosystems

Inland dunes in northwestern Europe support a number of dry vegetation types. These ecosystems are poor in nutrients and it has been suggested that accumulation of nutrients triggers succession in such systems.We studied the accumulation of organic matter and N and P over a 30 months period in two adjacent ecosystems, the Spergulo-Corynephoretum and the Genisto-Callunetum. Amounts of plant matter and soil organic matter significantly accumulated during the sampling period in the Genisto-Callunetum but not in the Spergulo-Corynephoretum. While nutrient concentrations of live and dead phytomass in the Spergulo-Corynephoretum were significantly higher than in the Genisto-Callunetum, total nutrient contents in the systems showed the opposite pattern. N and P concentrations in litter were relatively high compared with the other fractions of plant matter and the amount of N significantly increased in both ecosystems during the sampling period. Soil moisture contents showed a seasonal pattern. It was highest in the top soil layer and higher in the Genisto-Callunetum than in the Spergulo-Corynephoretum.The estimated annual increase of total N in these two ecosystems was consistent with rates of atmospheric N deposition (wet fall + dry fall) measured in comparable Dutch sites.