Savanna ecosystems in northern South America possess acid soils, low phosphorus (P) fertility, and low primary productivity. Dominant C 4 tussock grasses, such as Trachypogon plumosus , are the main contributor to total biomass production in neotropical savannas. Soil microbial activity, mineralization of organic P, and arbuscular mycorrhizal (AM) symbiosis are essential for understanding the adaptation of plants to the low P fertility. We selected a low P-sorbing Entisol and a high P-sorbing Vertisol, at Estación Experimental La Iguana located in the Orinoco Llanos in northeastern Venezuela, with T. plumosus as the dominant plant component. We monitored changes in organic P fractions, P transformation rates, dehydrogenase activity (DHA), acid phosphomonoesterase activity (APA), and AM status after P addition as a soluble phosphate salt, K 2 HPO 4 , or a slow release rock phosphate. Fertilizer addition resulted in a doubling of P-shoot content in plants growing in the Vertisol and no significant difference in the Entisol. Microbial activity, APA, and P transformation rates responded to P addition in the high P-sorbing Vertisol, but not in the low P-sorbing Entisol. Less available organic P fractions were affected by P addition from both sources. Although no differences in AM colonization were found, the high AM colonization demonstrates that T. plumosus functions as an obligate mycotroph. A deeper understanding of P cycling in the soil surrounding the roots of native savanna grasses will allow us to better respond to the anthropogenic pressures on these ecosystems.

Salt marshes are ecosystems of significant ecological importance for coastal stability and fundamental roles in marine ecosystems. Salt marshes are declining due to anthropogenic and natural causes including sea level rise. Coastal restoration efforts have increased worldwide, but many fail in long-term coastal stability. We used a naturally occurring arbuscular mycorrhizal fungus (AMF) to test whether survival and early growth of the salt marsh grass Sporobolus pumilus (formerly Spartina patens) improved under simulated salt marsh conditions. Using a tidal mesocosm bench, we grew inoculated plants with varying AMF treatments under simulated tidal regimes to determine if AMF could aid in establishment of healthy Sporobolus communities. Rhizome-derived S. pumilus had greater survival and grew faster than seed-derived plants. Plants inoculated with propagated AMF consistently outperformed both sterile and native sediment controls in terms of plant survival and growth. Use of rhizome-derived Sporobolus inoculated with propagated Funneliformis geosporum showed the most promise in producing successful plant populations for salt marsh restoration. This may be due to plant life stage and improved plant nutrient status, allowing rhizome-derived plants to grow more quickly than seed-derived plants. Using these plants in future large-scale restoration may increase re-establishment of salt marsh ecosystems.

We surveyed Spartina saltmarsh sediment rhizosphere fungal communities at three saltmarshes and two timepoints in coastal Nova Scotia. Based on ITS2 Illumina miSeq rDNA data and multivariate analysis, neither sediment zone nor collection period correlated with fungal ASV richness, but collection site did. However, Shannon diversity indicated that sediment zone played a significant role in fungal diversity. For unweighted and weighted UniFrac distance, site was the major factor driving beta-diversity, with sediment zone and collection period having smaller roles. Sediment type and saltmarsh plant species may play important roles in structuring rhizosphere fungal assemblages, here dominated by ascomycetes. To our knowledge, our study is the first to assess fungal sediment communities in saltmarshes in Atlantic Canada using metabarcoding. It provides a biodiversity analysis of sediment fungi in a poorly studied but highly important ecosystem and points to their roles in nutrient cycling, blue carbon, coastal stability and coastal restoration. Our work will inform ongoing saltmarsh restoration in Atlantic Canada.

Geum peckii (Rosaceae), the Eastern Mountain Avens, is a small herbaceous plant that is listed as endangered federally and provincially. In Canada, this species is found in bogs on Brier Island and Harris Lake, Digby County, Nova Scotia. The only other population outside of Canada is in New Hampshire (USA). To enhance conservation research of this species, a seed sample from the native species seed bank at Acadia University was used to establish a plant tissue culture of G. peckii plants. Survival of out-planted material was then assessed in both the greenhouse and the field. The field test site was within 20km of the existing plant populations in Digby County. Our study also revealed that G. peckii grows in association with arbuscular mycorrhizal fungi (AMF). During out-planting, plants received a mycorrhizal inoculum with the goal of enhancing survival. We used either a commercial mycorrhizal inoculum, or a native inoculum. Control plants were left untreated. Survival was 97-100 % among all the treatments by the end of the 2016 planting season. The results to date underscore the potential value of seed banking for protection of endangered native plant species. This study marks the first time in Nova Scotia that an endangered plant species has been successfully retrieved from seed bank storage, propagated by tissue culture, and out-planted back into a natural habitat. Keywords: Conservation, endangered native species, Geum peckii, tissue culture propagation

Saltmarshes are highly productive ecosystems that provide nursery and refuge habitat for animals, buffer storm-wave effects, and stabilize coastlines. Unfortunately, saltmarshes are in decline due to several cumulative stressors. Beneficial root-associated fungi are known to colonize >80% of land plants, but are understudied in intertidal zones. We examined arbuscular mycorrhizal fungi (AMF) in the roots of 2 dominant saltmarsh cordgrasses, Spartina patens (Saltmarsh Hay) and Spartina alterniflora (Smooth Cordgrass) (Poaceae), in the Minas Basin, NS, Canada. We collected 9 sediment cores at the beginning, middle, and end of the 2016 growing season (May-September) for each plant species (n = 54). We examined AMF root colonization using microscopy and fungal-DNA barcoding. Smooth Cordgrass had an AMF root colonization rate of 9%, while Saltmarsh Hay exhibited a higher AMF root colonization rate of 68%. We identified 1 AMF species, Funneliformis geosporum (Glomeraceae), in both host-plant species. We present the first Spartina spp. (cordgrasses) AMF root-colonization data for northeastern North America north of Connecticut, which may aid saltmarsh restoration efforts in Nova Scotia.

The distribution of microbial functional groups in soil may be governed by the interaction between the soil environment and the presence of other microbial competitors or facilitators. In forest soils, one of the most important groups of organisms are fungi, which are vital to many ecosystem processes such as nutrient cycling and decomposition, and can form direct connections to primary producers. Nevertheless, the overall effect of soil fungi on the structure and distribution of the other soil microbial functional groups has not been thoroughly investigated. We hypothesized that by altering the soil environment, fungi create favorable conditions for Archaea, methane oxidizing bacteria (MOB) and denitrifying bacteria (DNB), thereby potentially influencing the ability of forest soils to produce or consume greenhouse gases. To test these hypotheses, we studied the distribution of microbial functional groups and fungi in forest soil using molecular methods and related that distribution to soil environment and extracellular enzyme activity as a measure of microbial activity and metabolic effort. Non-metric multidimensional scaling of terminal restriction fragment length (TRFLP) profiles found that DNB and MOB largely separated within ordination space, suggesting little overlap of these bacteria in soil cores. In addition, DNB were significantly positively correlated with fungal biomass and with chitinase activity while MOB were negatively correlated with both. Most archaeal TRFs were also negatively correlated with fungal biomass, suggesting that forest Archaea and MOB have similar relationships to fungal biomass. Soil chemistry including soil carbon (C), nitrogen (N) and bicarbonate extractable phosphorus (P) were not significantly correlated with DNB, MOB or Archaea. Our results suggest that soil fungi might influence the spatial distribution of important prokaryotic groups in forests, including some groups that mediate the production and consumption of important greenhouse gases.

Plants and soil microbes produce extracellular enzymes (EE) that catalyze the hydrolysis of nitrogen (N) and phosphorus (P) containing compounds in soil and other enzymes involved in degradation of lignin and cellulose. We explored whether soil enzyme activity involved in carbon (C), N and P cycling were correlated with plant distribution, soil chemical conditions and the identity of fungi colonizing tree roots in an old growth forest remnant. Terminal restriction fragment length polymorphism (TRFLP) was used to determine the presence of root fungi and standard fluorometric analysis was used to determine soil enzyme activities. Soil enzymes were consistently positively correlated with soil C and N, but not CN ratio. Soil P was also correlated with enzyme activity during both June and September sampling. We saw no significant relationships between herbaceous plant cover and enzyme activity in June, but there were significant positive correlations between α-glucosidase and herbaceous plant coverage in September. We also found that some enzymes were significantly correlated with the identity of fungi colonizing tree roots separated from the soil cores. Chitinase and β-glucosidase were positively correlated with the genera Russula and Piloderma while chitinase was negatively correlated with Amanita and Entoloma. In addition, phosphatase was positively correlated with Russula, Meliniomyces and Solenopezia. Our results suggest that enzyme activity in old growth forest soils are affected by a variety of environmental factors, and that herbaceous plants and some root fungi may be associated with sites of elevated or decreased decomposition potential and nutrient cycling.

Forest litter is a large reservoir of organic compounds that adds CO2 to the atmospheric carbon pool when it decomposes. Predicting CO2 efflux from litter decomposition is difficult because litter can undergo significant diurnal and day-to-day shifts in temperature. Moreover, the relationship between temperature and respiration may change if the decomposer microorganisms acclimate to short-term temperature changes. Therefore, we studied the relationship between temperature and respiration by litter decomposer microorganisms in a Pinus resinosa (Ait.) system and tested the hypothesis that their respiration acclimates to temperature. We found only limited evidence for acclimation following 6 degrees C shifts for 7 days. This suggests that increase in respiratory CO2 loss associated with increased temperature would not be greatly ameliorated by physiological acclimation for periods of up to a week.

Minimally invasive surgery, which requires a different approach than used in the past, has created a revolution not only in surgical practice but also in surgical education. To overcome the challenges and difficulties of minimally invasive surgery, training needs to be done outside the operating room and away from the patient. New educational tools have emerged in the form of surgical simulators, including trainer boxes, virtual reality simulators and hybrid simulators. Many studies have confirmed the effectiveness of both box trainers and virtual reality simulators for surgical education. The integration of simulators into a structured laparoscopic skills curriculum creates an ideal training ground for acquiring the necessary skills for minimally invasive surgery. It has also been proven that this training model is effective for transferring acquired skills into the clinical setting.

ABSTRACT Although the level of diversity of root-associated fungi can be quite high, the effect of plant distribution and soil environment on root-associated fungal communities at fine spatial scales has received little attention. Here, we examine how soil environment and plant distribution affect the occurrence, diversity, and community structure of root-associated fungi at local patch scales within a mature forest. We used terminal restriction fragment length polymorphism and sequence analysis to detect 63 fungal species representing 28 different genera colonizing tree root tips. At least 32 species matched previously identified mycorrhizal fungi, with the remaining fungi including both saprotrophic and parasitic species. Root fungal communities were significantly different between June and September, suggesting a rapid temporal change in root fungal communities. Plant distribution affected root fungal communities, with some root fungi positively correlated with tree diameter and herbaceous-plant coverage. Some aspects of the soil environment were correlated with root fungal community structure, with the abundance of some root fungi positively correlated with soil pH and moisture content in June and with soil phosphorous (P) in September. Fungal distribution and community structure may be governed by plant-soil interactions at fine spatial scales within a mature forest. Soil P may play a role in structuring root fungal communities at certain times of the year.

Decomposer microorganisms contribute to carbon loss from the forest floor as they metabolize organic substances and respire CO2. In temperate and boreal forest ecosystems, the temperature of the forest floor can fluctuate significantly on a day-to-night or day-to-day basis. In order to estimate total respiratory CO2 loss over even relatively short durations, therefore, we need to know the temperature sensitivity (Q10) of microbial respiration. Temperature sensitivity has been calculated for microbes in different soil horizons, soil fractions, and at different depths, but we would suggest that for some forests, other ecologically relative soil portions should be considered to accurately predict the contribution of soil to respiration under warming. The floor of many forests is heterogeneous, consisting of an organic horizon comprising a few more-or-less distinct layers varying in decomposition status. We therefore determined at various measurement temperatures the respiration rates of litter, F-layer, and H-layer collected from a Pinus resinosa plantation, and calculated Q10 values for each layer. Q10 depended on measurement temperature, and was significantly greater in H-layer than in litter or F-layer between 5 and 17°C. Our results indicate, therefore, that as the temperature of the forest floor rises, the increase in respiration by the H-layer will be disproportionate to the increase by other layers. However, change in respiration by the H-layer associated with change in temperature may contribute minimally or significantly to changes of total forest floor respiration in response to changes in temperature depending on the depth and thickness of the layer in different forest ecosystems.

Despite the importance of ectomycorrhizal (ECM) fungi to both the carbon economy of their hosts (Smith & Read, 1997; Wallander et al., 2004) and ecosystem respiration (Rygiewicz & Andersen, 1994; Bååth & Wallander, 2003; Hobbie & Hobbie, 2006; Heinemeyer et al., 2007; Moyano et al., 2008), little is known about the factors that control their rates of respiration. We have recently found that substantial variation exists among ECM fungal isolates in the ability of respiration to acclimate to temperature (Malcolm et al., 2008). The potential for respiratory adaptation to temperature, an evolutionary response, has yet to be shown for ECM fungi. Therefore, in this study, we explored variation in respiration for ECM fungi in culture from two sites located at different latitudes. Fungi are presumed to adapt to prevailing temperatures because species taken from different climates exhibit temperature ranges for optimal growth, or exhibit distinct minimum or maximum temperatures for survival that correspond to their climates of origin (Cooke & Whipps, 1993; Tibbett et al., 1998; Robinson, 2001). The same is true for plants. With the presumption that temperature exerts a strong selective effect on respiration along environmental gradients (Reich et al., 1996), studies utilizing elevational or latitudinal gradients have shown repeatedly that plant respiration rates at a given measurement temperature are frequently higher for ecotypes from northern latitudes or higher elevations than for those from southern latitudes or lower elevations (Sowell & Spomer, 1986; Mariko & Koizumi, 1993; Reich et al., 1996). Therefore, we hypothesized that cultures of ECM fungi from near Fairbanks, Alaska would have higher respiration rates at a given measurement temperature than those from near State College, Pennsylvania, which, if found, would be suggestive of adaptation to temperature, as has been found for plants along environmental gradients. We also determined whether isolates from contrasting sites differed in respiratory sensitivity to temperature (Q10) in order to help predict whether the effects of future shifts in soil temperature on the respiration rates of ECM fungi depend on site. Because different fungal lineages have different evolutionary histories, it may not be valid to contrast an isolate of one lineage from higher latitude with another isolate of a different lineage from lower latitude (Burt, 1989). Thus, while we were not able to collect isolates of the same species from contrasting sites, we did make comparisons using congeneric contrasts. Specifically, we collected ECM fungi from four genera and three families: Amanitaceae (Amanita spp.), Russulaceae (Lactarius spp.) and Boletaceae (Leccinum spp. and Suillus spp.), each from both Alaska (latitude 65°07′N, longitude 147°30′) and Pennsylvania (latitude 40°48′N, longitude 77°54′) (Table 1). Fungi were cultured from field-collected sporocarps and maintained on a growth medium consisting of 19.5 g of potato dextrose agar (Difco; Becton, Dickinson & Co., Sparks, MD, USA), 7.5 g of Bacto Agar (Difco) and 0.375 g of NH4Cl per liter of water. Fungal cultures were initially maintained in incubators set to temperatures reflective of those experienced by the fungi during the growing season at their site of origin: 17°C for Pennsylvanian isolates and 11°C for Alaskan isolates (for more information about environmental temperatures, see Malcolm et al., 2008). Two isolates (one from Pennsylvania and one from Alaska) from each of the four genera were propagated using the growing medium described above in each of 10 100 × 20 mm Petri dishes, for a total of 80 dishes. Each isolate was initially grown at 11°C (Alaskan isolates) or 17°C (Pennsylvanian isolates) for 2–3 wk in order for each fungal colony to initiate new growth (c. 2–4 mm increase in diameter). After new growth had been established, half of the 10 dishes of each isolate were maintained in the initial incubator. The other half were shifted to the other incubator. Each of the replicate dishes was maintained for 1 wk in these incubators before respiration was assessed in order to allow for the potential for physiological acclimation to temperature (Malcolm et al., 2008). Steady-state respiration rates (CO2 exchange rates) were determined at 11, 17, and 23°C for each of the 80 dishes using a custom-built gas exchange system (Malcolm et al., 2008). In order to standardize respiration rates by fungal dry weight, fungal tissues were separated from agar by melting in test tubes held in boiling water immediately following the respiration measurements. The tissues were placed in a drying oven (60°C) until constant weight was achieved. The proportional change in respiration rate over a 10°C interval (Q10) was calculated by plotting the log-transformed respiration rates against measurement temperature, obtaining the slopes of the linear regression, and using the following equation (Atkin et al., 2000): Q 10 = 10(10 × slope) Because respiration rates were obtained across a range of measurement temperatures on the same set of individual cultures, we used the Repeated Measures Proc Mixed ANOVA Procedure in sas (version 9.1, 2002–2003; SAS Institute Inc., Cary, NC, USA) to analyze the respiration responses to temperature. We determined the effects of site, genus, measurement temperature and the interaction between site and measurement temperature on respiration rate when: isolates from Alaska were incubated at 11°C and isolates from Pennsylvania were incubated at 17°C; all isolates were incubated at a common temperature of 11°C; and all isolates were incubated at a common temperature of 17°C. To analyze Q10 we used the Proc GLM ANOVA Procedure in sas with site and genus as main effects. Differences were analyzed in the same cases as above. Irrespective of whether the isolates were incubated at temperatures reflective of their sites of origin (Alaskan isolates at 11°C and Pennsylvanian isolates at 17°C) or at common temperatures (either 11 or 17°C), the mean respiration rate was significantly higher at a given measurement temperature for isolates from Alaska than for isolates from Pennsylvania over the range 11–23°C (Fig. 1, Supporting information Table S1). The significant effect of site of origin was observed in all genera except for Amanita (Fig. 2). Furthermore, the average respiration rate of Alaskan isolates at a measurement temperature of 11°C (57.16 ± 4.17 µmol CO2 s−1 mg−1) was very close to that of Pennsylvanian isolates at a measurement temperature of 17°C (56.70 ± 4.17 µmol CO2 s−1 mg−1), irrespective of incubator temperature (Fig. 1). Mean (± 1 SEM) of respiration at three measurement temperatures for ectomycorrhizal fungi originating from Alaska and Pennsylvania incubated at 11 and 17°C. Mean (SEM) Q10 values are displayed for each site of origin at each incubation temperature. Q10 values followed by different uppercase letters are significantly different for the comparison of fungi from Alaska and Pennsylvania incubated at 11°C and 17°C, respectively; and Q10 values followed by different lowercase letters are significantly different for the comparison of fungi from both sites incubated at a common temperature of 11 or 17°C. n = 4 genera. Mean (± 1 SEM) of respiration, averaged over measurement temperatures of 11, 17 and 23°C, for each of four genera of fungi originating from Alaska and Pennsylvania. Fungi were incubated at 11 or 17°C. n = 5 Petri dishes for each combination of site × genus × treatment. These results are consistent with the hypothesis that the metabolism of ECM fungi can adapt to some factor related to site of origin. Because the two sites differ markedly in latitude, this factor may be temperature, but this is impossible to determine unequivocally because, in addition to temperature, vegetation type, soil chemistry and moisture may also differ between sites. Nevertheless, temperature differences along latitudinal gradients appear to create a strong selective pressure on respiration responses in plants (Reich et al., 1996), so it is possible that adaptation to temperature contributed at least partly to the observed phenomenon in ECM fungi. The large number of studies performed on plant latitudinal and altitudinal gradients with results that are consistent with adaptation to temperature (Sowell & Spomer, 1986; Mariko & Koizumi, 1993; Reich et al., 1996), as are ours, suggests that, if temperature is not the only factor to which organisms have adapted along such gradients, it may certainly be an important contributing factor. The Q10 values reported for the fungi in this study, between 1.82 and 2.05 for measurement temperatures between 11 and 23°C (Fig. 1), were close to the often-assumed value of 2.0 for most biological systems (Cox et al., 2000; Potter et al., 2001; Atkin et al., 2005). We have shown, however, that site may influence the sensitivity to temperature of ECM fungi. Overall, values of Q10 were significantly lower for isolates from Alaska than for isolates from Pennsylvania, except when the isolates were incubated at a common temperature of 11°C (Fig. 1, Table S2). Thus, as global temperatures increase, proportional increases in respiration for a given temperature shift could be lower for the fungi at high latitude than for those at lower latitude. Few comparable data exist for ECM fungi, but in studies of plants, Sowell & Spomer (1986) and Mariko & Koizumi (1993) indicated that no significant variation in Q10 was seen among ecotypes from different elevations. Further, the sensitivity of the respiration response to temperature is similar to that of roots, mycorrhizal fungal hyphae, and soil microbes, as previously shown by Bååth & Wallander (2003) in microcosm. We acknowledge that the respiration rates reported herein may not necessarily reflect absolute rates for fungi in symbio because of potential host effects on carbon supply. However, the respiration rates obtained in this study do agree with those obtained in previous studies of ECM fungi in culture (Taber & Taber, 1987; Souto et al., 2000; Malcolm et al., 2008), with those obtained in symbio (Rygiewicz & Andersen, 1994), and also with a field estimate calculated by Malcolm et al. (2008). Still, we urge modelers to use caution if utilizing data from this study, but to take note of the broader ecological pattern that we have shown. In summary, our results suggest that the metabolisms of ECM fungi from two widely divergent sites from varying latitudes may be inherently different which could, in turn, have far-reaching implications for the carbon economy of the host plant and ecosystem respiration. For example, if the host does have less control over carbohydrate transfer to the fungus than the fungus itself, then for plant hosts from lower latitudes, fungal isolates from higher latitudes may be inappropriate symbionts because of their disproportionately high respiration costs. Moreover, because ECM fungi contribute significantly to total soil respiration (Heinemeyer et al., 2007; Moyano et al., 2008), adaptation to prevailing temperatures would tend to equalize their contribution to total soil respiration rates along temperature gradients. Finally, our use of congeneric contrasts from a variety of families suggests a general propensity for physiological adaptation among the Basidiomycota. Further studies that include more latitudes and/or more replication within latitudes, and studies that consider the respiration response of ECM fungi in symbio, appear to be warranted. We thank B. Eissenstat, G. Laursen, L. Taylor, R. Ruess and T. Marr for collecting ECM sporocarps in Alaska. We are grateful to Jori Sharda, Josh Kucharski, Tom Adams, Tara Chrznowski, Upeksha Nanayakkara and Franz Lichtner for their help with various technical aspects of this project. We also thank Drs Richard Marini and Durland Shumway for their invaluable help with the statistical analyses. We greatly appreciate the constructive comments and suggestions of the anonymous reviewers on an earlier version of this manuscript. We acknowledge the United States National Science Foundation (IBN-0235403) for funding received by DME and RTK, the A. W. Mellon Foundation and the United States Department of Agriculture for funding received by RTK and the Pennsylvania State University College of Agriculture Competitive Grant program for funding received by GMM. Table S1 Analysis of variance of respiration of ectomycorrhizal fungi incubated at temperatures reflective of the litter of their sites of origin during the growing season (11°C for Alaskan isolates and 17°C for Pennsylvanian isolates) or at common temperatures (either 11 or 17°C). Table S2 Analysis of variance for the respiration Q10 value of four genera of fungi, each comprising two isolates, one from Alaska and one from Pennsylvania Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

G L E N N A M . M A L C O L M *, J U A N C . L Ó P E Z G U T I É R R E Z z, R O G E R T . K O I D E *w , a n d D AV I D M . E I S S E N S T AT *w *Intercollege Graduate Degree Program in Ecology, The Pennsylvania State University, University Park, PA 16802, USA, wDepartment of Horticulture, The Pennsylvania State University, University Park, PA 16802, USA, zThe Holden Arboretum/Case Western University, Kirtland, OH 44094, USA

Soil was amended with maize mucilage, a major rhizodeposit, to study its role on the number of culturable soil micro-organisms, the structure of the bacterial community, atrazine mineralization and atzC abundance. The maximal percentage of atrazine mineralization was lower for mucilage-amended than for water-amended soil. Total culturable soil bacteria and 16S rDNA copy number, measured by RT-PCR, presented similar values and were not significantly (P < 0.05) different among treatments. Mucilage applied at a rate of 70 microg C g(-1) dry soil day(-1) over two weeks did not modify the abundance of the total soil microflora. Global structure of soil bacterial communities revealed by RISA analysis was not modified by maize mucilage amendment. Abundance of atzC sequence was only augmented by mucilage addition at the beginning of the experiment. However, this increase was not sustainable in time, as atzC copy number increased in water-amended soil which, in turn, corresponded with the higher percentage of atrazine mineralization observed in this soil. Maize mucilage amendment alone contributed only to minor changes in the atrazine-degrading community in the studied soil.

Savanna ecosystems have low primary productivity, strong seasonality, and acid soils with low phosphorus (P) content. Organic P (Po) comprises around 50% of the total soil P and is plant-available only after mineralization. Rhizosphere processes mediated by plants, microorganisms and arbuscular mycorrhiza (AM) are important for plant P nutrition. We studied P transformation rates, Po-fractionation, acid phosphomonoesterase activity (APA), AM status, dehydrogenase activity (DHA), and bacterial and fungal plate counts in the rhizosphere of the native dominant grass Trachypogon plumosus. We collected samples from three acid savanna soils differing in order and P content (Entisol, Vertisol and Ultisol) at Estación Experimental La Iguana (Northeastern Venezuela) during the dry, rainy and transitional seasons over a 2-year-period. Less available Po fractions (moderately labile, moderately and highly resistant) seem to be involved in short-term P-cycling transformations as they significantly varied with season. During the rainy season plant P content (576–1160mgPkg−1 dry weight) and APA (44–200mgPNPkg−1 dry soil) were higher, while microbial number and activity (DHA) were lower. The higher P availability in the Entisol (6–9mgPkg−1 dry soil) resulted in a better plant nutritional status and inhibited APA. T. plumosus seems to be highly dependent on AM symbiosis (45–71% AM colonized root length, 0.6–8 AM spores g−1 dry soil), especially during the rainy season. Po mineralization processes, mediated by biological associations in the rhizosphere, are crucial for understanding seasonal P-cycling and fertility in acid savanna soils.

Nitrate reduction is performed by phylogenetically diverse bacteria. Analysis of narG (alpha subunit of the membrane bound nitrate reductase) trees constructed using environmental sequences revealed a new cluster that is not related to narG gene from known nitrate-reducing bacteria. In this study, primers targeting this as yet uncultivated nitrate-reducing group were designed and used to develop a real-time SYBR® Green PCR assay. The assay was tested with clones from distinct nitrate-reducing groups and applied to various environmental samples. narG copy number was high ranging between 5.08×108 and 1.12×1011 copies per gram of dry weight of environmental sample. Environmental real-time PCR products were cloned and sequenced. Data was used to generate a phylogenetic tree showing that all environmental products belonged to the target group. Moreover, 16S rDNA copy number was quantified in the different environments by real-time PCR using universal primers for Eubacteria. 16S rDNA copy number was similar or slightly higher than that of narG, between 7.12×109 and 1.14×1011 copies per gram of dry weight of environmental sample. Therefore, the yet uncultivated nitrate-reducing group targeted in this study seems to be numerically important in the environment, as revealed by narG high absolute and relative densities across various environments. Further analysis of the density of the nitrate-reducing community as a whole by real-time PCR may provide insights into the correlation between microbial density, diversity and activity.

Denitrification, the reduction of nitrate to nitrous oxide or dinitrogen, is the major biological mechanism by which fixed nitrogen returns to the atmosphere from soil and water. Microorganisms capable of denitrification are widely distributed in the environment but little is known about their abundance since quantification is performed using fastidious and time-consuming MPN-based approaches. We used real-time PCR to quantify the denitrifying nitrite reductase gene (nirK), a key enzyme of the denitrifying pathway catalyzing the reduction of soluble nitrogen oxide to gaseous form. The real-time PCR assay was linear over 7 orders of magnitude and sensitive down to 10(2) copies by assay. Real-time PCR analysis of different soil samples showed nirK densities of 9.7x10(4) to 3.9x10(6) copies per gram of soil. Soil real-time PCR products were cloned and sequenced. Analysis of 56 clone sequences revealed that all cloned real-time PCR products exhibited high similarities to previously described nirK. However, phylogenetic analysis showed that most of environmental sequences are not related to nirK from cultivated denitrifiers.

The impact of organic amendment (sewage sludge or waste water) used to fertilize agricultural soils was estimated on the atrazine-degrading activity, the atrazine-degrading genetic potential and the bacterial community structure of soils continuously cropped with corn. Long-term application of organic amendment did not modify atrazine-mineralizing activity, which was found to essentially depend on the soil type. It also did not modify atrazine-degrading genetic potential estimated by quantitative PCR targeting atzA, B and C genes, which was shown to depend on soil type. The structure of soil bacterial community determined by RISA fingerprinting was significantly affected by organic amendment. These results showed that modification of the structure of soil bacterial community in response to organic amendment is not necessarily accompanied by a modification of atrazine-degrading genetic potential or activity. In addition, these results revealed that different soils showing similar atrazine-degrading genetic potentials may exhibit different atrazine-degrading activities.