Louise E. Jackson

Application of Real-Time PCR To Study Effects of Ammonium on Population Size of Ammonia-Oxidizing Bacteria in Soil

ABSTRACT Ammonium oxidation by autotrophic ammonia-oxidizing bacteria (AOB) is a key process in agricultural and natural ecosystems and has a large global impact. In the past, the ecology and physiology of AOB were not well understood because these organisms are notoriously difficult to culture. Recent applications of molecular techniques have advanced our knowledge of AOB, but the necessity of using PCR-based techniques has made quantitative measurements difficult. A quantitative real-time PCR assay targeting part of the ammonia-monooxygenase gene (amoA) was developed to estimate AOB population size in soil. This assay has a detection limit of 1.3 × 105 cells/g of dry soil. The effect of the ammonium concentration on AOB population density was measured in soil microcosms by applying 0, 1.5, or 7.5 mM ammonium sulfate. AOB population size and ammonium and nitrate concentrations were monitored for 28 days after (NH4)2SO4 application. AOB populations in amended treatments increased from an initial density of approximately 4 × 106 cells/g of dry soil to peak values (day 7) of 35 × 106 and 66 × 106 cells/g of dry soil in the 1.5 and 7.5 mM treatments, respectively. The population size of total bacteria (quantified by real-time PCR with a universal bacterial probe) remained between 0.7 × 109 and 2.2 × 109 cells/g of soil, regardless of the ammonia concentration. A fertilization experiment was conducted in a tomato field plot to test whether the changes in AOB density observed in microcosms could also be detected in the field. AOB population size increased from 8.9 × 106 to 38.0 × 106 cells/g of soil by day 39. Generation times were 28 and 52 h in the 1.5 and 7.5 mM treatments, respectively, in the microcosm experiment and 373 h in the ammonium treatment in the field study. Estimated oxidation rates per cell ranged initially from 0.5 to 25.0 fmol of NH4+ h−1 cell−1 and decreased with time in both microcosms and the field. Growth yields were 5.6 × 106, 17.5 × 106, and 1.7 × 106 cells/mol of NH4+ in the 1.5 and 7.5 mM microcosm treatments and the field study, respectively. In a second field experiment, AOB population size was significantly greater in annually fertilized versus unfertilized soil, even though the last ammonium application occurred 8 months prior to measurement, suggesting a long-term effect of ammonium fertilization on AOB population size.

Short-term partitioning of ammonium and nitrate between plants and microbes in an annual grassland

We measured short-term (24 h) partitioning of 15NH4+ and 15NO3− into plants and microbes in a California annual grassland. Experiments were done in early spring at the peak of plant growth (February), and in late spring (April) when plant senescence had begun. Soil moisture decreased from 31 to 9% during this period. We injected either 15NH4+, 15NO3−, 15NH4+NO3− or NH415NO3− (≈ 2μg Ng−1 soil for each N-species) into the top 10cm of soil in cylinders which had been driven into the soil. After 24 h the soil and plants in the cylinders were harvested and we measured total N and 15N in inorganic forms, the microbial biomass (chloroform fumigation technique) and harvested plant material. Ammonium was the dominant source of N to both plants and microbes. In February, uptake rates of NH4+ were 81 and 426 mg N m−2 day−1 for plants and microbes, respectively, while in April the rates were 110 and 639 mg N m−2 day−1. Rates of NO3− uptake were 41 and 81 mgNm−2day−1 in February and 83 and 146mgNm−2 day−1 in April for plants and microbes, respectively. Thus, microbes took up substantially more NH4+ and NO3− than plants. On both sampling dates, NH4+ concentrations in the top 10cm of soil ranges from 600 to 800mg N m−2, andNO3− concentrations were <50 mg N m−2 High rates of microbial NO3−3 uptake may have resulted from the occurrence of microsites that were depleted in NH4+. Even though plants competed better for NO3− than for NH4+, microbial uptake was a major factor controlling NO3− availability to plants. The high rates of NH4+ and NO3− uptake by plants and microbes clearly demonstrate that the soil N pool bears little relationship to actual N availability.

Soil microbial community composition and land use history in cultivated and grassland ecosystems of coastal California

Phospholipid ester-linked fatty acid (PLFA) profiles were used to evaluate soil microbial community composition for 9 land use types in two coastal valleys in California. These included irrigated and non-irrigated agricultural sites, non-native annual grasslands and relict, never-tilled or old field perennial grasslands. All 42 sites were on loams or sandy loams of similar soil taxa derived from granitic and alluvial material. We hypothesized that land use history and its associated management inputs and practices may produce a unique soil environment, for which microbes with specific environmental requirements may be selected and supported. We investigated the relationship between soil physical and chemical characteristics, management factors, and vegetation type with microbial community composition. Higher values of total soil C, N, and microbial biomass (total PLFA) and lower values of soil pH occurred in the grassland than cultivated soils. The correspondence analysis (CA) of the PLFA profiles and the canonical correspondence analysis (CCA) of PLFA profiles, soil characteristics, and site and management factors showed distinct groupings for land use types. A given land use type could thus be identified by soil microbial community composition as well as similar soil characteristics and management factors. Differences in soil microbial community composition were highly associated with total PLFA, a measure of soil microbial biomass, suggesting that labile soil organic matter affects microbial composition. Management inputs, such as fertilizer, herbicide, and irrigation, also were associated with the distinctive microbial community composition of the different cultivated land use types.

Spatial and temporal effects on plant-microbial competition for inorganic nitrogen in a California annual grassland

The changes in N-dynamics which occur after the start of autumn rains, following an extended summer drought, were examined in a California annual grassland. Competition for NH+4and NO−3 between plants and microbes, and the role of spatial compartmentalization. were studied using short-term ( < 24 h) experiments using 15N pool dilution and tracer techniques. Temporal dynamics of mineralization, nitrification and simultaneous plant and microbial uptake of NH4+ and NO−3 were assessed in intact soil microcosms periodically watered to simulate autumn rains. During the first week after initial soil wetting, both mineralization and immobilization rates increased; mineralization rate continued to increase during the next 6 weeks but immobilization rate remained constant. After 6 weeks of simulated wet-season, microbes consumed more of both NH+4 and no−3 than did plants in 8 h diurnal 15N tracer experiments. Though ambient NO−3 concentrations were low, nitrification accounted for about 13 of the N mineralized. Plants competed better for NO−3 than for NH4+. This suggests that nitrification benefitted plants by increasing accessible N. Spatial factors were critical in controlling N-dynamics. Microsites free of NH+4 were presumably responsible for the extensive microbial NO3− uptake. Of the activities measured in the top 9cm of the soil, the surface 5mm accounted for almost half of mineralization and plant NH4+-uptake, but only 11% of microbial NH4+-uptake.

Wet-dry cycles affect dissolved organic carbon in two California agricultural soils

Abstract During Californias hot, dry summers, irrigated soils are subjected to frequent wet–dry cycles and surface layers dry to near air–dry conditions between irrigations. We investigate whether wet–dry cycles enhance soil dissolved organic carbon (DOC) concentrations. This research follows up on previous observations of higher DOC concentrations in the surface (0–2 cm) than deeper (2–15 cm) soil layer late in the growing season, even when soils were moist throughout the profile. We also investigate whether DOC contents correspond to other measures of C available to microorganisms. All measurements were made on soils stabilized at −0.03 MPa water potential for 48 h at 25°C to avoid the initial pulse of microbial activity which follows re-wetting of dry soils. After 3 months during the summer field season, DOC concentrations increased 2.5-fold in the surface 0–2 cm layer and 1.20 to 1.35-fold in the 2–15 cm layer in soils under both organic (N inputs of cover crop and manure) and conventional (inorganic N inputs) management for irrigated tomatoes. In microcosms exposed to wet–dry cycles for 3 months, DOC concentrations increased by 70%, while in microcosms maintained at −0.03 MPa for 3 months DOC remained constant. The increase in DOC in both field and microcosm soils exposed to wet–dry cycles indicates that wet–dry cycles contribute to higher background DOC contents. The greater DOC increase in the field than microcosms may be due to evaporation causing upward movement of water and concentrating DOC at the soil surface, or to greater C availability in the field due to the presence of plant roots. Respiration and microbial biomass C (MBC) remained constant or declined slightly in both soil layers and microcosm treatments over the growing season, counter to the trends in DOC concentration. Therefore DOC contents measured under moist soil conditions do not appear to consistently indicate C availability to microorganisms. The percentage of labile DOC, as measured by a bioassay, declined in the surface layer of the organic field soil and in organic and conventional soils in both microcosm treatments over the 3 month experiment, possibly indicating that roots were a continuing source of labile DOC in the lower field layers. Reflecting the higher organic inputs to the organic than conventional soil, DOC, MBC and respiration rates were 2–2.5 times higher in the organic than conventional soil throughout the experiments, however the percentage of labile DOC was approximately twice as high in the conventional soil as in organic soil.

Rapid response of soil microbial communities from conventional, low input, and organic farming systems to a wet/dry cycle

Soil microbial communities may be strongly influenced by agricultural practices which change the soil environment. One such practice is the use of organic amendments and cover crops which increase carbon availability to microorganisms. Another is irrigation which, in California’s hot, rain-free growing season, can cause severe wet/dry cycles. We investigated (i) long-term diAerences in amounts of organic inputs using soils from organic, low input, and conventional farming systems, and (ii) diAerences in severity of soil drying following irrigation, using soil from two depths, 0‐3 and 3‐15 cm. All soils were air-dried and re-wetted, and we measured short-term changes in microbial biomass carbon (MBC), dissolved organic carbon (DOC), respiration, and phospholipid ester-linked fatty acid (PLFA) composition before and for 27 h after re-wetting. Respiration rates were fit to a two-first-order-component model. Carbon respired from the more slowly utilized C pool of the two-component model, MBC, and DOC increased with increasing amounts of organic inputs, and PLFA composition of the organic and conventional soils clearly diAered in their mole percentages of numerous fatty acids when analyzed by principal components analysis and redundancy analysis. Despite these diAerences, the response of microbial communities in the three farming systems to soil drying and re-wetting was similar. For example, the relative increase in MBC following soil re-wetting did not diAer among the farming system soils. In contrast, the relative increase in MBC after re-wetting was greater, and the respiratory response to soil re-wetting was more rapid in the surface (0‐3 cm) than deeper (3‐15 cm) layer. Higher ratios of cyclopropyl fatty acids to their precursors suggested greater stress to bacteria in the deep than surface layer, and these ratios declined more rapidly after re-wetting in the deep than surface layer. This study suggested that adaptation to wet/dry cycles by surface microorganisms had occurred during the 3-month growing season, leading to changes in both microbial process rates and community composition. # 1999 Published by Elsevier Science Ltd. All rights reserved.

Changes in microbial biomass and community composition, and soil carbon and nitrogen pools after incorporation of rye into three California agricultural soils

Abstract The effects of long-term agricultural management on active soil organic matter (SOM) and short-term microbial C and N dynamics were investigated. Short-term changes in chemical and biological variables after incorporating fresh rye shoots were measured in intact soil cylinders from three contrasting agricultural systems. Two of the soils were from organic or conventional 4-yr rotations which had been in place for 6 yr as part of the University of California at Davis Sustainable Agriculture Farming Systems (SAFS) project and the third was from a double-cropped, intensive vegetable production system in the Salinas Valley of California. Microbial biomass (MB) and respiration, numbers of organisms in several trophic groups, soil inorganic N, dissolved organic C and recoverable rye were measured before and during the 6 weeks following rye incorporation. Active soil organic matter, expressed as the ratios of microbial biomass C or N to total soil C or N, respectively, appeared to be related to long-term management. These ratios increased in proportion to increased organic inputs and reduced tillage or periods of fallow. In all soils, MBC increased and decreased rapidly following rye incorporation, but MBN was fairly constant. Significant differences among the soils in MBC and MBN were maintained over the 6 week experiment. Following rye incorporation, fluorescein diacetate (FDA) active counts of bacteria and bacterial-feeding nematodes increased rapidly, whereas changes in FDA active fungal hyphal lengths and fungal-feeding nematodes were less pronounced. The rates of rye decomposition, respiration and net N mineralization were highest the first week after incorporation, coincident with increases in MBC and numbers of active bacteria in all three soils. There were significant differences among soils in numbers of organisms in the trophic groups on some sample dates, but changes in soil respiration and inorganic N and the rate of rye decomposition remained similar in all three soils. The SAFS organic soil had a somewhat lower ratio of bacterial to fungal biomass and lower ratio of respiration to MBC throughout the experiment than the SAFS conventional soil. Despite long-term differences in agricultural management and differences in active SOM contents among the three soils, the rates of rye decomposition and C and N mineralization were similar. Rye incorporation produced a short-term burst of microbial growth and activity of similar magnitude in all three soils although the initial MB contents in the three soils were different. Variations among the soils in FDA active counts of fungi and numbers of bacterial- and fungal-feeding nematodes indicated that microbial community composition was more responsive to rye incorporation than were changes in soil C and N pools.

Microbial responses to simulated tillage in cultivated and uncultivated soils

Tillage is known to have long-term eAects on organic matter and labile pools of nutrients in soil, but the short-term changes in microbial dynamics and activity after tillage are less well understood. We investigated the immediate eAects of simulated tillage on microbial community structure as determined by phospholipid fatty acid (PLFA) profiles, microbial activity, and carbon (C) and nitrogen (N) pools. Intact cores were obtained from Chualar sandy loam soils under grassland and vegetable production. The top 15 cm of soil was sieved to simulate tillage, then the cores were incubated in the greenhouse. Sampling took place 1 day before the tillage simulation and throughout the next 2 weeks. In the grassland soil, multivariate analysis showed changes in PLFA profiles within hours, indicating rapid changes in microbial community structure. Specific PLFA markers indicated a reduction in microeukaryotic biomass as well as an increase in a microbial stress marker after sieving. Respiration (as determined by soil incubation in sealed containers) decreased immediately after sieving and continued to decline through the next 14 days. Sieving was followed by a continuous accumulation of nitrate. In the vegetable soil, the changes in PLFA profiles were slow and gradual. The PLFA stress indicator rose only slightly. Microbial activity and biomass were low, and only small changes occurred in most variables. A decline in respiration and an increase in nitrate occurred several days after sieving. In both soils, decreased soil moisture may have contributed to changes in soil responses after sieving. Short-term responses to tillage may be less pronounced in soils with a long history of cultivation because of a relatively resilient microbial community and/or because lower initial microbial biomass and nutrient pools preclude a strong response to disturbance. 7 2000 Elsevier Science Ltd. All rights reserved.

Arbuscular mycorrhizas, microbial communities, nutrient availability, and soil aggregates in organic tomato production

Effects of arbuscular mycorrhzal (AM) fungi on plant growth and nutrition are well-known, but their effects on the wider soil biota are less clear. This is in part due to difficulties with establishing appropriate non-mycorrhizal controls in the field. Here we present results of a field experiment using a new approach to overcome this problem. A previously well-characterized mycorrhizal defective tomato mutant (rmc) and its mycorrhizal wildtype progenitor (76R MYC+) were grown at an organic fresh market tomato farm (Yolo County, CA). At the time of planting, root in-growth cores amended with different levels of N and P, were installed between experimental plants to study localized effects of mycorrhizal and non-mycorrhizal tomato roots on soil ecology. Whilst fruit yield and vegetative production of the two genotypes were very similar at harvest, there were large positive effects of colonization of roots by AM fungi on plant nutrient contents, especially P and Zn. The presence of roots colonized by AM fungi also resulted in improved aggregate stability by increasing the fraction of small macroaggregates, but only when N was added. Effects on the wider soil community including nematodes, fungal biomass as indicated by ergosterol, microbial biomass C, and phospholipid fatty acid (PLFA) profiles were less pronounced. Taken together, these data show that AM fungi provide important ecosystem functions in terms of plant nutrition and aggregate stability, but that a change in this one functional group had only a small effect on the wider soil biota. This indicates a high degree of stability in soil communities of this organic farm.

Plant and soil nitrogen dynamics in California annual grassland

Seasonal changes in soil water and nitrogen availability were related to the phenology and growth of plants in California annual grassland. Plant accumulation of nitrogen was mainly confined to two short periods of the year: fall and early spring. At these times, plants were in the vegetative growth phase, roots were growing rapidly and soil moisture was high. During these periods, soil nitrate was low or depleted. High flux of nitrogen in this ecosystem, however, is indicated by the rapid disappearance of the previous years detrital material, high microbial biomass, and high mineralizable nitrogen and nitrification potential.At the end of the summer drought, significant amounts of the previous years detrital material had disappeared, chloroform-labile N (expressed as microbial biomass N) was at its seasonal maximum, and soil inorganic nitrogen pools were high. This suggests inorganic nitrogen flux during the drought period. The ‘drought escaper’ life history characteristics of annual grasses in California annual grassland, however, may prevent plants from utilizing available nitrogen during a large part of the year.