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Measuring soil nitrogen (N) provides important information for ecosystem productivity and improving N use efficiency in agricultural systems. Conventional means of sampling N using soil extractions disturb soil structure and function, and likely distort accurate quantification. In situ microdialysis is a novel sampling method that generates differing N profiles compared to soil extractions. Here we test the hypothesis that differences observed between sampling methods are due to the minimal disturbance and sampling of a mobile N fraction when using microdialysis, with discernible patterns expected across soils with distinct clay and organic matter contents. In a short-term laboratory microcosm experiment with 21 sugarcane cropping soils, we compared salt (potassium chloride; KCl) or aqueous (H2O) extractants and microdialysis. KCl-extractable ammonium (NH4+) was highly correlated with the content of clay, total N and carbon, indicative of bound N being solubilised. In contrast, NH4 (+) contributed significantly less to microdialysis fluxes and was not correlated with the measured soil properties, which we attribute to minimal disturbance of bound N center dot H2O extracts sampled proportionally more NH4 (+) than microdialysis but were significantly correlated with fluxes. This suggests that while microdialysis and H2O extraction sample from a dissolved N pool, H2O extracts sample from an additional pool of loosely-bound NH4+. Nitrate (NO3) measures were correlated between methods, but shared no relationship with the measured soil properties, indicating that NO3 sampling is less affected by the disturbance introduced by extractions. We conclude that sampling inorganic N is biased by the degree to which soil sampling methods disturb adsorbed N sources with implications for interpreting soil N measurements.

Delays between soil sampling and processing for analysis are common in both research and agronomy, but the effects of storage conditions on measurements of plant-available nitrogen (N) are rarely considered. With increasing recognition of organic N pools in soils, such as amino acids and peptides, it is necessary to determine how sample handling impacts the outcomes of soil N quantification. In this study, we used in situ microdialysis to approximate plant availability of amino acids, ammonium and nitrate, then compared to both potassium chloride (KCl) extract and microdialysis samples taken from excavated soil samples when in the field, after 24 h refrigerated storage, and after storage for 1 month, either refrigerated or air-dried. Nitrate levels measured with microdialysis and KCl extracts increased immediately after soil sampling and continued to accumulate in the next day and 1 month stored samples. Amino acid and ammonium measurements remained more constant; however, microdialysis showed a decline in amino acid-N between in situ and next day samples. The proportional representation of N pools in the in-field extracts was most similar to in situ microdialysis. Soil samples should be processed for N analysis as close to sampling as possible, and the storage duration and conditions reported. The influence of storage must be considered in interpreting soil test results.

Ameliorating biological attributes of agricultural soils is desirable, and one avenue is introducing beneficial microbes via commercial biostimulant products. Although gaining popularity with farmers, scientific evaluation of such products in field-grown crops is often lacking. We tested two microbial products, Soil-Life™ and Nutri-Life Platform®, in a commercial sugarcane crop by profiling bacterial and fungal communities in soil and roots using high throughput phylogenetic marker gene sequencing. The products, one predominantly consisting of Lactobacillus and the other of Trichoderma, were applied as a mixture as per manufacturers’ instructions. Additives included in the formulations were not listed, and plots that did not receive the product mixture were the controls. The compositions of bacterial communities of soil and sugarcane roots, sampled 2, 5 and 25 weeks after application, were unaffected by the products. Soil fungal communities were also unaffected, but sugarcane roots had a greater relative abundance of three unidentified taxa in genera Marasmius, Fusarium and Talaromyces in the treated plots. Sugarcane yield was similar across all treatments that included a 25% lower nitrogen fertiliser rate. Further research must examine if the altered root fungal community is a consistent feature of the tested products, and if it conveys benefits. We conclude that putative biostimulants can be evaluated by analysing the composition of microbial communities. DNA profiling should be complemented by cost-benefit analysis to build a public information base documenting the effects of microbial biostimulants. Such knowledge will assist manufacturers in product development and farmers in making judicious decisions on product selection, to ensure that the anticipated benefits of microbial biostimulants are realised for broad acre cropping.

Widespread and continuing losses of tropical old-growth forests imperil global biodiversity and alter global carbon (C) cycling. Soil organic carbon (SOC) typically declines with land use change from old-growth forest, but the underlying mechanisms are poorly understood. Ecological restoration plantations offer an established means of restoring aboveground biomass, structure and diversity of forests, but their capacity to recover the soil microbial community and SOC is unknown due to limited empirical data and consensus on the mechanisms of SOC formation. Here, we examine soil microbial community response and SOC in tropical rainforest restoration plantings, comparing them with the original old-growth forest and the previous land use (pasture). Two decades post-reforestation, we found a statistically significant but small increase in SOC in the fast-turnover particulate C fraction. Although the δ13C signature of the more stable humic organic C (HOC) fraction indicated a significant compositional turnover in reforested soils, from C4 pasture-derived C to C3 forest-derived C, this did not translate to HOC gains compared with the pasture baseline. Matched old-growth rainforest soils had significantly higher concentrations of HOC than pasture and reforested soils, and soil microbial enzyme efficiency and the ratio of gram-positive to gram-negative bacteria followed the same pattern. Restoration plantings had unique soil microbial composition and function, distinct from baseline pasture but not converging on target old growth rainforest within the examined timeframe. Our results suggest that tropical reforestation efforts could benefit from management interventions beyond re-establishing tree cover to realize the ambition of early recovery of soil microbial communities and stable SOC. Electronic supplementary material The online version of this article (10.1007/s00248-019-01414-7) contains supplementary material, which is available to authorized users.

Soil extracellular enzyme activity (EEA) represents a critical bottleneck in the release of bioavailable nutrients from organic materials. However, quantifying spatial and temporal dynamics of EEA remains challenging. Techniques which measure the activity of, or directly sample free enzymes in situ may assist in understanding the short-term exoproteomic responses of microbes and roots to substrates, but few tools exist to explore EEA with minimal disturbance. We explore the potential of in situ microdialysis to directly sample soil enzymes, measuring their activity using a modified enzyme assay. We hypothesise that the technique's bias towards free solutes will also allow differentiation of free and stabilised enzyme pools. As little is known about the efficiency of microdialysis to sample enzymes from soil, recovery of a protease standard was quantified from solution and soil, finding that enzyme recovery is hindered at lower soil moisture contents. We further measured the response of native protease activity after the addition of soybean litter to clay and sandy soils, finding microdialysis observed greater EEA in litter-amended treatments than controls in both soil types. In comparison, EEA as measured by conventional extraction-incubation methods was only greater in amended clay soils. In a final experiment, hydrolytic enzyme activity of free and stabilised clay soil fractions were estimated using microdialysis. Free enzymes contributed 9% of total hydrolytic activity in soil without litter, increasing to 46% in litter-amended soil, suggesting fresh litter promoted a transient increase in the production of free exoenzymes by soil microbes. In contrast, the addition of litter had no significant effect on stabilised EEA. In spite of the obvious challenges involved in applying microdialysis as a method for soil protein sampling, this method offers new possibilities for investigating challenging spatial and temporal aspects of enzyme dynamics and protein availability in soils.

Soil incubation studies commonly use archived, dry stored soil samples. Numerous studies have detected a profound impact of dry storage on soil microbial community composition and activity. However, comparatively few studies have explored the impact of storage on the short-term mineralisation of soil organic matter (SOM) after moistening dry soils to revive and stabilise microbial activity (“pre-incubation”). We examined whether pre-incubation of dry stored soil of contrasting texture (Alfisol and Vertisol) can recover native soil microbial functions in fresh soil or soil stored for 3 weeks, 3 or 36 years. The oldest samples had a delayed CO2 response after moistening, but matched CO2 production rates of younger samples within hours. The two soil types displayed contrasting CO2 emission responses after moistening; the Alfisols had a rapid but short-lived CO2 emission peak (the so-called “Birch Effect”) while the Vertisols demonstrated a long, sustained release of CO2 reflecting its higher abundance of microsite abundance and microaggregates which may protect SOM and microorganisms during adverse conditions like drying. After 10 days of pre-incubation, older Alfisol samples had higher protease activity and response to amino acid addition demonstrating altered microbial physiological responses relating to nitrogen (N) compared to fresher counterparts. However, such responses in Vertisols were unchanged suggesting high clay soils with high potential microsite abundance may improve preservation of soil N functions through dry storage or even drought. This work highlights both limitations and possibilities of incubating dry archived soils for SOM mineralisation studies.

Nutrient-rich organic wastes and soil ameliorants can benefit crop performance and soil health but can also prevent crop nutrient sufficiency or increase greenhouse gas emissions. We hypothesised that nitrogen (N)-rich agricultural waste (poultry litter) amended with sorbents (bentonite clay or biochar) or compost (high C/N ratio) attenuates the concentration of inorganic nitrogen (N) in soil and reduces emissions of nitrous oxide (N2O). We tested this hypothesis with a field experiment conducted on a commercial sugarcane farm, using in vitro incubations. Treatments received 160 kg N ha−1, either from mineral fertiliser or poultry litter, with additional N (2–60 kg N ha−1) supplied by the sorbents and compost. Crop yield was similar in all N treatments, indicating N sufficiency, with the poultry litter + biochar treatment statistically matching the yield of the no-N control. Confirming our hypothesis, mineral N fertiliser resulted in the highest concentrations of soil inorganic N, followed by poultry litter and the amended poultry formulations. Reflecting the soil inorganic N concentrations, the average N2O emission factors ranked as per the following: mineral fertiliser 8.02% &gt, poultry litter 6.77% &gt, poultry litter + compost 6.75% &gt, poultry litter + bentonite 5.5% &gt, poultry litter + biochar 3.4%. All emission factors exceeded the IPCC Tier 1 default for managed soils (1%) and the Australian Government default for sugarcane soil (1.25%). Our findings reinforce concerns that current default emissions factors underestimate N2O emissions. The laboratory incubations broadly matched the field N2O emissions, indicating that in vitro testing is a cost-effective first step to guide the blending of organic wastes in a way that ensures N sufficiency for crops but minimises N losses. We conclude that suitable sorbent-waste formulations that attenuate N release will advance N efficiency and the circular nutrient economy.

Delivering nutrients from mineral or organic fertilizers out of synchrony with crop uptake causes inefficiencies and pollution. We explore methodologies for evaluating sorbents as additives to organic agricultural wastes to retain nitrogen in an exchangeable form and deliver at rates that approximate the uptake capacity of roots. Focussing on ammonium (NH4+) as the main inorganic nitrogen form in the studied wastes (sugarcane mill mud, poultry litter), we tested geo-sorbents and biochar for their ability to retain NH4+. Sorption capacity was ranked palagonite

Soil organic matter (SOM) is crucial to soil health, supporting most of the soil properties that benefit plant growth and ecosystem services including carbon sequestration, nutrient recycling and water infiltration. Recent study is exposing the soil microbial community as not only decomposing SOM, but also providing the primary source of chemicals for its formation. All else equal, SOM formation is theoretically greatest when microbes maximise enzyme efficiency (ratio of enzyme activity to carbon loss from respiration) and biomass efficiency (biomass gain per unit substrate added). Our study examines the relationship between microbial composition and metrics of these two efficiencies. We hypothesised that both will increase with higher ratios of soil fungi to bacteria. We manipulated microbial composition through sustained use of selective microbial inhibitors in microcosms with tropical soil, alongside variation of litter quality and diversity and the presence or absence of a simulated root exudate. Both litter and inhibitor treatments significantly changed soil microbial composition and function, and enzyme efficiency and biomass efficiency were both higher in microbial communities with more fungi, supporting our hypothesis. Structural equation modelling suggested that the observed efficiency changes did indeed occur in part via changes in microbial composition after accounting for direct effects of treatments. Taken together the results provide some support for the hypothesis that soil fungi benefit SOM formation.

Root exudates are thought to promote nitrogen (N) availability via rhizosphere interactions, but empirical evidence is difficult to obtain given the scale and temporary nature of these processes. Microdialysis has potential to simulate root exudation patterns and quantify the effects on N availability simultaneously, but this has so far not been attempted. In a conceptual root exudation study, we used sucrose as a simple C source to investigate if microdialysis could detect the effects of continuous localised C supply on soil inorganic N fluxes. Through retrodialysis we released sucrose and simultaneously monitored diffusive soil N fluxes over one week, followed by a further seven days without sucrose. Based on current understanding of rhizosphere N dynamics, we hypothesised that N fluxes are inversely related to sucrose release, and upon ceasing release, N fluxes would increase. Using a 5 mM sucrose perfusate, C releases resulted in decreased N fluxes, but contrary to our hypothesis, N fluxes did not increase after ceasing sucrose release (c.f. control soil). Diffusive sucrose efflux from microdialysis probes increased in soils amended with N-rich litter suggesting that microbial activity and associated sucrose consumption altered sucrose concentration gradients. The fluxes of sucrose breakdown products glucose and fructose were greatest in litter treatments receiving sucrose, indicative of increased invertase activity in the presence of fresh organic matter. In the short term (days), sucrose release did not prompt an increase in inorganic N availability, possibly because of stimulated microbial growth and increased N demand under C-rich conditions. Our study confirms that microdialysis allows time-sensitive insight into the dynamic interactions of carbon and N in the rhizosphere.

Microdialysis is a technique that can be used to sample fluxes of nitrogen (N) in soils with minimal disturbance. To advance our understanding of the technique and improve N recovery, we compared a common membrane type (10 x 0:5 mm probe length and width, 20 kDa molecular weight cut-off; MWCO) with alternative length and MWCO configurations (30 mm; and 100 kDa MWCO). We hypothesised that the alternative membranes would improve recovery of low molecular weight N via increased surface area and membrane pore size. The test environments, sampled at fixed pump flow rates, were: (i) stirred 100 mu M N standard solution containing organic (amino acid) and inorganic (ammonium, nitrate) N; (ii) soil spiked with 100 mu M standard N solution; and (iii) in situ boreal forest soil. In general, long membranes recovered more N, but the magnitude of improved recovery varied with test environment. Long membranes recovered more inorganic N regardless of flow rate, except ammonium in stirred solution, where length had no effect at slow flow rates. Long membranes also recovered more organic N from stirred solution regardless of flow rate, and recovered most N at slow flow rates in spiked soil. Longer membranes recovered more amino acids in situ in forest soil, with improved resolution of individual amino acids, but were biased towards soluble, mobile forms. MWCO did not affect N recoveries, indicating that in the test conditions, membrane length had greater control than pore size. We discuss the bottlenecks of microdialysis application in soil research and conclude that optimised membrane configurations will advance its use as a tool for quantifying nutrient fluxes in soils. (C) 2017 Elsevier Ltd. All rights reserved.

Plant root exudates are believed to increase root capture of nutrients (including nitrogen) by encouraging development of a rhizosphere root community, and providing them with an energy source to facilitate degradation of litter and soil organic matter. However, observing the consequences of root exudation on nutrient cycling and microbial activity is challenging with current methods, given the small scales involved. We investigated the effect of root exudation on nitrogen (N) availability by simulating root exudation with microdialysis. This novel technique enables continuous release of synthetic solutions of root exudates via diffusion in situ in soil by using a root-sized permeable membrane. Importantly, it also allows for simultaneously monitoring the effects on inorganic N fluxes. To emulate growth of a root tip through a specific soil region, sucrose was released for seven days before substituting sucrose with water for a further 7 days. We investigated boreal forest soils with and without litter amendments (ground pea shoots) to attain different C/N ratios and we used two rates of exudation by retrodialysing with either 0.5 or 5 mM sucrose solution. We observed that pea litter promoted significant N immobilisation, along with greater rates of sucrose release from microdialysis probes - peaking at 90.7 ± 8 µg sucrose m-2 s-1 using the 5 mM sucrose solution after three days. This suggests that greater root exudation may be driven by microbial demand for both C and N, with no short-term nutritional benefit for plant roots, even after exudation has ceased within a specific soil region. Glucose and fructose fluxes (breakdown products of sucrose) were also greatest in the litter treatment, indicating enzyme activity was promoted by the availability of both sucrose and litter. CO2 respiration measurements indicated significant differences between litter and control soils, but there was no detectable effect of sucrose exudation, suggesting that the small amounts of C supplied and the limited area influenced by the diffusion of sucrose had little impact on overall microcosm respiration. We conclude that short-term C exudation presented no immediate benefit for plant nutrition in our experiment. Future studies can benefit from using microdialysis to investigate the influence of more complex root exudate solutions, as well as the mechanistic roles of transpiration-induced mass flow on plant N availability in the rhizosphere.

This study sought to identify whether piggery effluent-derived nitrogen sources can be formulated with urea and nitrification inhibitors to better synchronize nitrogen (N) supply with crop demand than conventional urea fertilizer alone. A 288 pot pasture growth and leaching growth accelerator trial (5 pasture cuts) was completed with a factorial treatment structure of three N sources (2.63 g N [kg soil]−1 applied as 100% urea-N, 8% struvite-N + 92% urea-N, and 8% piggery pond sludge-N + 92% urea-N), five rates of three nitrification inhibitors (including 3,4-Dimethylpyrazole phosphate, DMPP; limonene+ethanol; and dicyandiamide, DCD), and matrix encapsulated forms of these inhibitors. Applying a combination of piggery sludge with urea increased N uptake during the first 4 weeks of plant growth (by 65%), though total N uptake throughout the trial (22 weeks) did not differ across the N-sources. The microbial community of the soil to which the sludge was added was significantly different from the un-amended soil at the conclusion of the trial. All inhibitor formulations significantly decreased leaching losses of mineral-N relative to the control (by 14 to 61%). The use of DMPP decreased initial nutrient uptake, deferring uptake until later in the experiment. Inhibitor addition resulted in microbial community effects that persisted throughout the trial. The study demonstrated that a piggery-derived N-source and a nitrification inhibitor can be used to manipulate plant N uptake to occur later or earlier in a growing period with equal cumulative uptake, achieving an 11% increase in residual N store, and decreased N leaching losses.

Microdialysis is emerging as a sensitive tool in environmental sciences, allowing for in situ sampling of solutes with minimal disturbance to soil environments. This perspective presents the theoretical foundations and practical applications of the technique with a focus on its use in soil, microbial and plant sciences. Using small probes (usually 0.5 mm diameter) fitted with permeable membranes, soil solutes are sampled, so that solute flux (and potentially concentration) can be calculated at root-relevant scales. However, physical and biological characteristics of soil systems impose challenges to sampling efficiencies with microdialysis, particularly where solute concentrations are low. Experimental parameters such as perfusate flow rates, probe design and sampling times can also influence the findings, and reduce the comparability between studies. We explore how equipment setup and experimental conditions can be optimised for soil environments, and how standardisation and calibration techniques may improve cross-study comparability. We show that the technique's strength lies in the ability to integrate many soil factors into a biologically relevant measure of solute availability. Microdialysis has so far provided new insight regarding the bioavailability of soil nitrogen (N) and phosphorus (P), where fluxes have been related to root uptake rates and gaseous fluxes from soil. The technique has also been used as a root simulator, mimicking exudation of organic acids to mobilise soil P, and to measure the potential contribution of transpiration-induced mass flow on N availability at the root surface. With further development, and paired with sensitive analytical methods and equipment, microdialysis has much potential to explore fragile and dynamic soil processes in biologically-active zones such as the rhizosphere, and could contribute to solving challenges relating to under- and oversupply of nutrients to plants. We conclude that, with further advancements and critical evaluation, microdialysis could become an important instrument in the soil analysis toolkit.

Improvement in fertilizer use efficiency is a key aspect for achieving sustainable agriculture in order to minimize costs, greenhouse gas emissions, and pollution from nutrient run-off. To optimize root architecture for nutrient uptake and efficiency, we need to understand what the roots encounter in their environment. Traditional methods of nutrient sampling, such as salt extractions can only be done at the end of an experiment, are impractical for sampling locations precisely and give total nutrient values that can overestimate the nutrients available to the roots. In contrast, microdialysis provides a non-invasive, continuous method for sampling available nutrients in the soil. Here, for the first time, we have used microCT imaging to position microdialysis probes at known distances from the roots and then measured the available nitrate and ammonium. We found that nitrate accumulated close to roots whereas ammonium was depleted demonstrating that this combination of complementary techniques provides a unique ability to measure root-available nutrients non-destructively and in almost real time.

Soil microbial communities and their activities are altered by land use change; however impacts and extent of these alterations are often unclear. We investigated the functional responses of soil microbes in agricultural soil under sugarcane and corresponding native soil under Eucalyptus forest to additions of contrasting plant litter derived from soybean, sugarcane and Eucalyptus in a microcosm system, using a suite of complimentary techniques including enzyme assays and community level physiological profiles (CLPP). Initially agricultural soil had 50% less microbial biomass and lower enzyme activities than forest soil, but significantly higher nitrification rates. In response to litter addition, microbial biomass increased up to 11-fold in agricultural soil, but only 1.8-fold in forest soil, suggesting a prevalence of rapidly proliferating 'r' and slower growing 'K' strategists in the respective soils. Litter-driven change in microbial biomass and activities were short lived, largely returning to pre-litter addition levels by day 150. Decomposition rates of sugarcane and soybean litter as estimated via CO2 production were lower in agricultural than in forest soil, but decomposition of more recalcitrant Eucalyptus litter was similar in both soils, contradicting the notion that microbial communities specialise in decomposing litter of the dominant local plant species. Enzyme activities and community level physiological profiles (CLPP) were closely correlated to microbial biomass and overall CO2 production in the agricultural soil but not the forest soil, suggesting contrasting relationships between microbial population dynamics and activity in the two soils. Activities of enzymes that break down complex biopolymers, such as protease, cellulase and phenol oxidase were similar or higher in the agricultural soil, which suggests that the production of extracellular biopolymer-degrading enzymes was not a factor limiting litter decomposition. Enzyme and CLPP analyses produced contrasting profiles of microbial activity in the two soils; however the combination of both analyses offers additional insights into the changes in microbial function and community dynamics that occur after conversion of forest to agricultural land.

Addressing the limited knowledge of nitrogen (N) pools in tropical agricultural soils and the need to reduce N losses from these systems, we analysed soluble organic and inorganic N in two Hydrosols under sugarcane. Concentrations of ammonium and nitrate spanned similar to 3 - orders of magnitude (0.2-41.0 mg ammonium-N, 0-10.7 mg nitrate-N kg(-1) soil) with the highest concentrations detected within 2-3 months of fertiliser application. Soluble amino acids spanned 1-order of magnitude (0.22-2.42 mg amino acid-N kg(-1) soil) and accounted for up to 70% of the low-molecular weight N. Amino acid concentrations were usually highest in the wet season and uniform across soil depth, indicating that amino acids are generated throughout the studied profile. We compared soluble and dissolved (free) N in the soil solution in a subset of samples. In soil solution, amino acid, ammonium and nitrate concentrations averaged 20,265 and 1820 mu M, respectively, corresponding to similar to 10% (amino acids), similar to 20% (ammonium) and similar to 100% (nitrate) of the soluble N pool. We calculated an annual gross amino acid flux in the dissolved N pool in the order of 2-6 tons N ha(-1) yr(-1) in the upper 40 cm of soil. We discuss whether amino acids can significantly contribute to the N demand of sugarcane. (C) 2012 Elsevier B.V. All rights reserved.

Fire is an important driver of nutrient cycling in savannas. Here, we determined the impact of fire frequency on total and soluble soil nitrogen (N) pools in tropical savanna. The study sites consisted of 1-ha experimental plots near Darwin, Australia, which remained unburnt for at least 14 years or were burnt at 1-, 2- or 5-year intervals over the past 6 years. Soil was analysed from patches underneath tree canopies and in inter-canopy patches at 1, 12, 28, 55 and 152 days after fire. Patch type had a significant effect on all soil N pools, with greater concentrations of total and soluble (nitrate, ammonium, amino acids) N under tree canopies than inter-canopy patches.The ‘time since the last fire’ had no significant effect on N pools. Fire frequency similarly did not affect total soil N but it did influence soluble soil N. Soil amino acids were most prominent in burnt savanna, ammonium was highest in infrequently burnt (5-year interval) savanna and nitrate was highest in unburnt savanna.We suggest that the main effect of fire on soil N relations occurs indirectly through altered tree-grass dynamics. Previous studies have shown that high fire frequencies reduce tree cover by lowering recruitment and increasing mortality. Our findings suggest that these changes in tree cover could result in a 30% reduction in total soil N and 10–60% reductions in soluble N pools. This finding is consistent with studies from savannas globally, providing further evidence for a general theory of patchiness as a key driver of nutrient cycling in the savanna biome.

Cotton cropping systems in Australia have poor nitrogen (N) use efficiency, largely due to over-application of N fertiliser. The N mineralisation from soil organic N reserves is often overlooked, or underestimated despite recent studies indicating that it may contribute the majority of N exported with the crop. Predicting N mineralisation is a major challenge for agricultural industries worldwide, as direct measurements are time-consuming and expensive, but there is considerable debate as to the most reliable methods for indirect estimation. Additionally, laboratory incubations assess potential (rather than actual) mineralisation, and may not be representative of N cycling rates in the field. We collected 177 samples from most major Australian cotton growing regions, and assessed their mineralisation potential using ex situ laboratory incubations, along with an assessment of potential indicators routinely measured in soil nutrient tests. Additionally, at three unfertilised sites we conducted in situ assessment of mineralisation by quantifying soil N at the beginning of the growing season, and soil and crop N at the end of the season. We found that Australian cotton cropping soils had substantial mineralisation potential, and that soil total N and total carbon were correlated with mineralisation, and have potential to be used for prediction. Other potential indicators such as carbon dioxide production and ammonium and nitrate concentrations were not correlated with mineralisation. In parallel studies of ex situ and in situ mineralisation, we found ex situ laboratory incubations overestimated mineralisation by 1.7 times on average. We discuss findings in terms of management implications for Australian cotton farming systems.

Globally only ≈50% of applied nitrogen (N) fertilizer is captured by crops and the remainder can cause pollution via runoff and gaseous emissions. Synchronizing soil N supply and crop demand will address this problem, however current soil analysis methods provide little insight into delivery and acquisition of N forms by roots. We used microdialysis, a novel technique for in situ quantification of soil nutrient fluxes, to measure N fluxes in sugarcane cropping soils receiving different fertilizer regimes and compare these with N uptake capacities of sugarcane roots. We show that in fertilized sugarcane soils, fluxes of inorganic N exceed the uptake capacities of sugarcane roots by several orders of magnitude. Contrary, fluxes of organic N closely matched roots’ uptake capacity. These results indicate root uptake capacity constrains plant acquisition of inorganic N. This mismatch between soil N supply and root N uptake capacity is a likely key driver for low N efficiency in the studied crop system. Our results also suggest that (i) the relative contribution of inorganic N for plant nutrition may be overestimated when relying on soil extracts as indicators for root-available N and (ii) organic N may contribute more to crop N supply than is currently assumed.

Worldwide, land use change from forest to intensive cropping agriculture impacts on soil quality, fertility and health, and can lead to decreased agricultural productivity over time. Such land use change typically results in changed structure and reduced quantities of soil microbial communities and an altered nitrogen (N) cycle. In soil of natural ecosystems, microbial breakdown and mineralisation of organic matter are main drivers of the N cycle, while in intensive cropping soils the N cycle is strongly supplemented by applications of N fertiliser. Applications of N fertiliser rates that generally exceed crop N needs are intended to safeguard yields, but often result in N losses from soil, sustaining negative effects on other ecosystems, greenhouse gas budgets and farm costs. In Australia, sugarcane is typically grown as an intensive monoculture, and in many regions has been cultivated almost continually since land was cleared of native forest. Australian sugarcane production is typified by high N applications and low crop N recovery that averages ~40% of applied fertiliser. Despite the observed high N losses from sugarcane cropping systems, detailed knowledge of soil N and carbon (C) dynamics is missing. This knowledge gap was addressed here to provide impetus for future improvements of N dynamics in sugarcane production. We studied N cycle dynamics and microbial function in sugarcane soil, spanning from litter decomposition, to pools and fluxes of organic N (protein and amino acids), and production rates and pool sizes of inorganic N (ammonium and nitrate). To place sugarcane soil in context, we compared soil from a nearby forest remnant to allow discerning changes in microbial function and soil N dynamics that have occurred as a result of land use change. Further investigations centred around the effects of C addition (as plant litter or sucrose) on microbial function and N cycling. Following findings that amino acids are an unexpectedly prevalent N form in sugarcane soils, we examined amino-N pools and fluxes using conventional soil extractions and novel in-situ microdialysis. Compared to forest soil, sugarcane soils had decreased microbial biomass, quantities of N in litter and amino acid pools, and increased rates of nitrification and nitrate concentrations. The capacity of soil microbial communities to decompose litter was examined in controlled conditions and showed that forest soil had a higher capacity for litter degradation. In response to litter addition, microbial biomass in forest soil remained steady, while microbial biomass in sugarcane soil increased up to 10-fold. Microbial enzyme production was higher in forest soil under most conditions. Addition of sucrose resulted in greater increases in respiration in sugarcane than forest soils. Overall, forest and sugarcane soil accommodated contrasting N and C cycles with negligible N2O emission and substantial methane uptake in forest soil, and considerable N2O emissions and no discernible methane uptake in sugarcane soil. Nitrogen fertiliser addition and high ammonification and nitrification rates resulted in ammonium and nitrate dominating the pool of low molecular weight N compounds (LMW), in sugarcane soil early in the cropping season (1-3 months post fertiliser application). Thereafter LMW-N concentrations remained low for the remainder of the year. During this period, amino acids represent a substantial proportion of the LMW-N pool. High turnover rates of amino acids result in large fluxes of N passing through this pool. These results indicate significant changes in microbial functional ecology and N cycling as a result of land use change. Sugarcane soils are typified by decreased microbial biomass, but an increase in microbial species which rapidly respond to substrates that can be easily metabolised, such as amino acids and sugars. The high nitrification rate that typified all fertilised sugarcane studied here, including N derived from synthetic urea and organic sources, has implications for N losses from soil. Nitrate ions are highly mobile and are prone to leaching or denitrification, potentially exacerbated by a preference of sugarcane for ammonium over nitrate. Sugarcane soil under best practice management (the lnew farming systemr) showed some improvement with respect to increased microbial biomass over conventional management; however both systems have similar N cycles. The high prevalence of amino acids in N pools and diffusive fluxes of sugarcane soil in mid to late growing season suggests that amino acids may play a role for crop N nutrition. Together, the findings here provide insights into soil and microbial processes that can assist the design of future sugarcane farming systems with increased N use efficiency and reduced N loss. These systems will need to make better use of ecosystem services provided by soil microbes, therefore knowledge of microbial processes and nutrient dynamics will be of increasing importance over coming decades.n

Modern agriculture is based on the notion that nitrate is the main source of nitrogen (N) for crops, but nitrate is also the most mobile form of N and easily lost from soil. Efficient acquisition of nitrate by crops is therefore a prerequisite for avoiding off-site N pollution. Sugarcane is considered the most suitable tropical crop for biofuel production, but surprisingly high N fertilizer applications in main producer countries raise doubt about the sustainability of production and are at odds with a carbon-based crop. Examining reasons for the inefficient use of N fertilizer, we hypothesized that sugarcane resembles other giant tropical grasses which inhibit the production of nitrate in soil and differ from related grain crops with a confirmed ability to use nitrate. The results of our study support the hypothesis that N-replete sugarcane and ancestral species in the Andropogoneae supertribe strongly prefer ammonium over nitrate. Sugarcane differs from grain crops, sorghum and maize, which acquired both N sources equally well, while giant grass, Erianthus, displayed an intermediate ability to use nitrate. We conclude that discrimination against nitrate and a low capacity to store nitrate in shoots prevents commercial sugarcane varieties from taking advantage of the high nitrate concentrations in fertilized soils in the first three months of the growing season, leaving nitrate vulnerable to loss. Our study addresses a major caveat of sugarcane production and affords a strong basis for improvement through breeding cultivars with enhanced capacity to use nitrate as well as through agronomic measures that reduce nitrification in soil.

Non-mycorrhizal Hakea actites (Proteaceae) grows in heathland where organic nitrogen (ON) dominates the soil nitrogen (N) pool. Hakea actites uses ON for growth, but the role of cluster roots in ON acquisition is unknown. The aim of the present study was to ascertain how N form and concentration affect cluster root formation and expression of peptide transporters. Hydroponically grown plants produced most biomass with low molecular weight ON> inorganic N>high molecular weight ON, while cluster roots were formed in the order no-N>ON>inorganic N. Intact dipeptide was transported into roots and metabolized, suggesting a role for the peptide transporter (PTR) for uptake and transport of peptides. HaPTR4, a member of subgroup II of the NRT1/PTR transporter family, which contains most characterized di- and tripeptide transporters in plants, facilitated transport of di- and tripeptides when expressed in yeast. No transport activity was demonstrated for HaPTR5 and HaPTR12, most similar to less well characterized transporters in subgroup III. The results provide further evidence that subgroup II of the NRT1/PTR family contains functional di- and tripeptide transporters. Green fluorescent protein fusion proteins of HaPTR4 and HaPTR12 localized to tonoplast, and plasma- and endomembranes, respectively, while HaPTR5 localized to vesicles of unknown identity. Grown in heathland or hydroponic culture with limiting N supply or starved of nutrients, HaPTR genes had the highest expression in cluster roots and non-cluster roots, and leaf expression increased upon re-supply of ON. It is concluded that formation of cluster roots and expression of PTR are regulated in response to N supply.

Soil microbial activity can be constrained by availability of energy because soil carbon (C) occurs mostly as complex soil organic matter (SOM), with relatively small quantities of high-energy, labile C. Decomposition of SOM is mediated by energy-requiring processes that need extracellular enzymes produced by soil microbial communities. We examined how an increase in energy status via sucrose supplementation affects the production of SOM-degrading enzymes, comparing matched soils under forest and sugarcane agriculture with histories of contrasting inputs of complex and labile C. Activities of SOM-degrading enzymes increased in both soils after sucrose addition, but CO2 production increased more rapidly in the sugarcane soil. The forest soil had greater increases in phosphatase and glucosidase activities, whereas the sugarcane soil had greater increases in protease and urease activity. The contrasting microbial community-level physiological profiles of the soils further diverged at 30 and 61 days after sucrose amendment, before returning to near pre-treatment profiles by 150 days. We interpreted the increasing soil enzyme production as indicative that enzyme production was limited by energy availability in both soils, despite contrasting histories of labile v. recalcitrant C supply. Quicker responses in sugarcane soil suggest pre-selection towards populations that exploit labile inputs.

Organic forms of nitrogen (ON) represent potential N sources for crops and an alternative to inorganic N (IN, ammonium nitrate). Sugarcane soils receive organic harvest residues (~40–100 kg ON ha–1), but it is unknown whether ON is a direct N source for crops. We investigated whether sugarcane can use organic monomers in the form of amino acids and whether the use of amino acids as a N source results in distinct metabolic or morphological change when compared with use of inorganic N (IN). Plantlets cultivated in sterile culture and young plants grown in non-sterile soil culture were supplied with IN, ON (five amino acids present in sugarcane soils), or combined IN and ON. All treatments resulted in similar biomass and N content indicating that sugarcane has a well developed capacity to use ON and confirms findings in other species. ON-supplied plants in axenic culture had increased total branch root length per unit primary root axis which has not been reported previously. In both experimental systems, ON supplied plants had increased asparagine concentrations suggesting altered N metabolism. Root of ON-supplied soil-grown plants had significantly reduced nitrate concentrations. We interpret the shift from nitrate to asparagine as indicative of N form use other than or in addition to nitrate by sugarcane. N metabolite profiling could advance knowledge of crop N sources and this will aid in development of N efficient cropping systems with a reduced N pollution footprint.

While importance of amino acids as a nitrogen source for plants is increasingly recognised, other organic N sources including small peptides have received less attention. We assessed the capacity of functionally different species, annual and nonmycorrhizal Arabidopsis thaliana (L.) Heynh. (Brassicaceae) and perennial Lobelia anceps L.f. (Campanulaceae), to acquire, metabolise and use small peptides as a N source independent of symbionts. Plants were grown axenically on media supplemented with small peptides (2–4 amino acids), amino acids or inorganic N. In A. thaliana, peptides of up to four amino acid residues sustained growth and supported up to 74% of the maximum biomass accumulation achieved with inorganic N. Peptides also supported growth of L. anceps, but to a lesser extent. Using metabolite analysis, a proportion of the peptides supplied in the medium were detected intact in root and shoot tissue together with their metabolic products. Nitrogen source preferences, growth responses and shoot–root biomass allocation were species-specific and suggest caution in the use of Arabidopsis as the sole plant model. In particular, glycine peptides of increasing length induced effects ranging from complete inhibition to marked stimulation of root growth. This study contributes to emerging evidence that plants can acquire and metabolise organic N beyond amino acids.