15N Natural Abundance Research Articles

Co-existence of Rhizobia and Diverse Non-rhizobial Bacteria in the Rhizosphere and Nodules of Dalbergia odorifera Seedlings Inoculated with Bradyrhizobium elkanii, Rhizobium multihospitium–Like and Burkholderia pyrrocinia–Like Strains

Rhizobia induce root nodules and fix atmospheric N2 for most legume species in exchange for carbon. However, the diverse endophytic non-rhizobial bacteria in legume nodules that co-exist with rhizobia are often ignored because they are difficult to cultivate using routine cultivation approaches. To enhance our understanding of the incidence and diversity of legume–bacteria associations, a high-throughput sequencing analysis of bacterial 16S rRNA genes was used to examine the bacterial community in the rhizospheres and root nodules of Dalbergia odorifera seedlings that were uninoculated or inoculated with Bradyrhizobium elkanii H255, Rhizobium multihospitium–like HT221, or Burkholderia pyrrocinia–like H022238, in two growth media (nitrogen [N]-supplied soil or N-omitted potting mix). Seedlings inoculated with Bradyrhizobium had significantly more nodules than seedlings in the other inoculation conditions, regardless of growth media. Using the 15N natural abundance method, it was shown that the inoculated plants had significantly higher N2 fixation efficiency (48–57%) and specific nodule activity [269–313 μg N mg−1 of dry weight (dwt) nodule] compared to the uninoculated plants (203 μg N mg−1 dwt nodule). The 16S rRNA gene analysis showed that there was generally a higher bacterial diversity in the rhizosphere than in the nodules in the corresponding condition. Both rhizobial inoculation and media status significantly altered the bacterial communities in the rhizospheres and nodules (P < 0.05), with the exception of the inoculated soil rhizospheres. Regarding non-rhizobial bacteria, three genera, i.e., Lactococcus, Bacillus, and Pseudomonas, were consistently enriched in the rhizosphere and Bradyrhizobium, Chloroplast norank (which belongs to Cyanobacteria), and Lactococcus were commonly found in the nodules. In contrast, common rhizobial genera (including Rhizobium, Mesorhizobium, and Burkholderia) were only present in the nodules at low relative abundances (0.01–3.41%). Regarding non-rhizobial bacteria, 32 genera were found in the nodules, with non-rhizobial bacteria being predominant in the N omitted potting mix (with a relative abundance of 56–87%). This study suggests that legume nodules are inhabited by a high diversity of non-rhizobial bacteria, which may play a vital role in nodulation and N2 fixation in the host plants.

Selecting elite groundnut (Arachis hypogaea L) genotypes for symbiotic N nutrition, water-use efficiency and pod yield at three field sites, using 15N and 13C natural abundance

About 70% of the groundnut (Arachis hypogaea L.) produced in Ghana is from the Guinea savanna. However, low soil nutrients, especially N, together with erratic rainfall distribution have often resulted in poor grain yield. The aim of this study was to evaluate plant growth, N2-fixing efficiency, N contribution, water-use efficiency and pod yield of 21 elite groundnut genotypes in the Guinea savanna of Ghana, using the 15N natural abundance technique. The data revealed significant variations in plant growth, symbiotic N contribution, and pod yield among the 21 genotypes tested at each field site. Average N contribution by groundnut genotypes ranged from 48 to 108 kg N ha−1. Also, mean pod yield ranged from 0.58 to 2.1 t ha−1. Genotypes ICGV-IS 08837, ICG 6222, ICGV 03315 and NKATIESARI demonstrated superior plant growth, symbiotic N contribution and greater pod yield. In fact, ICGV-IS 08837 yielded almost 2.5 fold more than CHINESE which is the most widely cultivated variety in the region. Genotypes ICGV-IS 08837, ICG 6222, ICGV 03315 and ICGV 99247 are therefore recommended for development into varieties for the Guinea savanna of Ghana. Genotypes ICG (FDRS) 4, ICGV00362 and ICGV99247 exhibited increased water-use efficiency, but were low in N2 fixation and N contribution, and would therefore be good parental material in breeding programs aimed at enhancing water-use efficiency in high N2-fixing genotypes.

Depth trends of soil organic matter C:N and 15N natural abundance controlled by association with minerals

Plant residues show carbon:nitrogen (C:N) decreases, 15N isotopic enrichment and preferential loss of labile substrates during microbial decay. In soil profiles, strikingly similar patterns of decreasing C:N and 15N isotopic enrichment with increasing depth are well documented. The parallel trend in organic matter composition with soil depth and during plant residue decay has been used as evidence to suggest that organic products accumulate or develop in the subsoil due to increasing intensity of microbially-driven processing, although no studies to date have verified this. Here, by applying sequential density fractionation, specific surface area, oxalate extractable Fe and Al, C:N and δ15N measures with depth to soils with relatively uniform soil mineralogy (Oxisols), climates and vegetation we show that changes in organo-mineral associations drive subsoil C:N and δ15N and C:N depth patterns more than in situ organic matter decay. Our results provide the first direct evidence that soil depth trends could be driven by mineral association instead of in situ processing.

Seasonal variation in natural abundance of δ13C and 15N in Salicornia brachiata Roxb. populations from a coastal area of India

ABSTRACTHigh and fluctuating salinity is characteristic for coastal salt marshes, which strongly affect the physiology of halophytes consequently resulting in changes in stable isotope distribution. The natural abundance of stable isotopes (δ13C and δ15N) of the halophyte plant Salicornia brachiata and physico-chemical characteristics of soils were analysed in order to investigate the relationship of stable isotope distribution in different populations in a growing period in the coastal area of Gujarat, India. Aboveground and belowground biomass of S. brachiata was collected from six different populations at five times (September 2014, November 2014, January 2015, March 2015 and May 2015). The δ13C values in aboveground (−30.8 to −23.6 ‰, average: −26.6 ± 0.4 ‰) and belowground biomass (−30.0 to −23.1 ‰, average: −26.3 ± 0.4 ‰) were similar. The δ13C values were positively correlated with soil salinity and Na concentration, and negatively correlated with soil mineral nitrogen. The δ15N values of aboveground (6.7–16.1 ‰, average: 9.6 ± 0.4 ‰) were comparatively higher than belowground biomass (5.4–13.2 ‰, average: 7.8 ± 0.3 ‰). The δ15N values were negatively correlated with soil available P. We conclude that the variation in δ13C values of S. brachiata was possibly caused by soil salinity (associated Na content) and N limitation which demonstrates the potential of δ13C as an indicator of stress in plants.

Exploration of Biological Markers of Feed Efficiency in Young Bulls.

The efficiency with which ruminants convert feed to desirable products is difficult to measure under normal commercial settings. We explored the use of potential biological markers from easily obtainable samples, that is, blood, hair, and feces, to characterize potential causes of divergent efficiency when considered as residual feed intake (RFI) or feed conversion efficiency (FCE). A total of 54 Charolais bulls, 20 in period 1 and 34 in period 2, were examined for individual dry matter intake (DMI) and growth. Bulls were offered a diet of 70:30 wrapped grass silage to concentrate for 99 d. At the conclusion of the test period, blood samples were collected for the determination of vitamins B2 and B6, and plasma used for the determination of metabolites, natural isotopic 15N abundance (15N NIA, expressed as δ15N ‰) and fractionation (Δ15Nplasmaproteins-diet and Δ13Cplasmaproteins-diet) and near-infrared spectroscopy (NIRS). Feces were analyzed by NIRS. Bulls were slaughtered at 15-17 months of age and carcass characteristics determined. Bulls were ranked according to RFI with extremes (SD ± 0.5; n = 31) classified as either efficient (Neg-RFI) or inefficient (Pos-RFI). Extreme bulls were then classified for FCE (high vs low FCE), changing the groups. Pos-RFI bulls consumed 14% more feed than Neg-RFI bulls for the same level of weight gain. Low FCE bulls tended to eat more, but had lower weight gains than high FCE bulls. No differences were detected in carcass conformation, fat scores, hot carcass weight, or dressing percentage. Yet, heart and bladder weights were heavier in Pos-RFI, and rumen weight tended to be heavier in Pos-RFI bulls. RFI did not affect bulk 15N or 13C fractionation. A negative correlation was observed between FCE and Δ15Nplasmaproteins-diet. Inefficient bulls (Pos-RFI) had higher δ15N in glycine compared to Neg-RFI bulls. Similarly, metabolomic analysis showed a tendency for concentrations of glycine and sarcosine to be elevated in Pos-RFI bulls, whereas aspartic acid and carnosine tended to be elevated, and serine tended to be lower in High FCE. Among vitamins, only flavin adenine dinucleotide concentration was higher in the blood of bulls with High FCE. These results suggest that the two feed efficiency metrics differ in the underlying mechanisms of metabolism, where RFI is driven by differences in the energetic requirements of visceral organs and the extent of AA catabolism.

Compost Legacy Down‐Regulates Biological Nitrogen Fixation in a Long‐Term Field Experiment

Core Ideas Compost‐based management enhanced soil organic matter in a Michigan Alfisol. Nitrogen fixation rates in soybean and red clover varied with management history. Weather markedly influenced the quantity of N fixed in three crop systems. Soil labile pool influence on N fixation can act as an internal regulator. Biological nitrogen fixation (BNF) is a fundamental process relied on in agriculture, yet few field studies have examined regulation through soil inorganic N feedbacks or considered seasonal effects. In a Michigan long‐term field experiment we examined soil labile C and N pools and impact on BNF in two species, over multiple years. The 15N natural abundance method was used to quantify BNF, with nodulated and non‐nodulated soybean [Glycine max (L.) Merr.] isolines and red clover (Trifolium pratense L.). Soil organic C status of plots was consistent with a gradient established through historical inputs: 0.8% (fertilizer management) to 0.9% (+ cover crop) to 1.2% (+ compost + cover crop). The fraction of nitrogen derived from atmosphere (fNdfa) in red clover and soybean over 3 yr was 61 to 58% with compost management, and 71 to 72% with fertilizer. This represented a downregulation of 19% (red clover) and 15% (soybean) in compost and cover crop managed plots, relative to inorganic fertilizer. In addition to management effects, we found that weather markedly influenced the total amount of N fixation over the 3 yr of the study. A mesic season supported vigorous soybean growth and 195 kg N ha−1 BNF, compared to 95 and 90 kg N ha−1 BNF in a dry and an excessively wet season. This study found that compost‐based management increased pools of labile C and N that internally downregulated BNF, while enhancing soybean yield compared to conventional management.

Effects of thinning on nitrogen status of a larch plantation, illustrated by 15N natural abundance and N resorption

ABSTRACTLarch (Larix spp.) is widely distributed in the boreal and temperate areas. Nitrogen (N) is considered as the major limiting element for these areas. Thinning is a common forest management practice. Thus, it is imperative to obtain a better understanding on how thinning could affect N status of larch plantations, and thus optimize the thinning intensity for sustainable forest management. In this study, we measured N concentrations and 15N natural abundance (δ15N) of needles and surface soil (0–10 cm) in a larch plantation with T0, T25 and T50 treatments (0%, 25% and 50% thinning intensities, respectively) in Northeast China. We found that needle and soil δ15N in T25 was the lowest, and the highest in T50. No significant differences were observed for needle and soil N concentrations among the thinning intensities. T25 exhibited the highest N resorption efficiency, indicating highest N use efficiency. Overall, N cycling in T25 was more closed than the control, and with lower soil N availability, while N cycling in T50 was more opened. Our study indicates that foliar 15N natural abundance is sensitive to thinning and can be potentially used to optimize thinning intensity from the perspective of N cycling.

Yield of sugarcane varieties and their sugar quality grown in different soil types and inoculated with a diazotrophic bacteria consortium

Sugarcane is a crop of great economic, social, and environmental relevance in Brazil. The country is the largest sugar producer and the second largest bioethanol producer in the world. The goal of this study was to evaluate the efficiency of a sugarcane inoculant composed of five diazotrophic bacterial strains, as well as nitrogen fertilization of two sugarcane varieties. Two experiments were carried out on two varieties using an experimental design composed of complete randomized blocks in a factorial of two varieties and three treatments with four replicates. The treatments can be described as: inoculation with the consortium of five diazotrophic strains, or N fertilization with 120 kg ha−1, and one control treatment. The following parameters were then evaluated: stem yield, accumulation of total dry matter, nitrogen content, quality of the sugarcane juice, and 15N natural abundance on flag-leaves. Inoculation and N fertilization on the Sapucaia plantation promoted increases of stem yield equivalent to 22.3 and 26.5 Mg ha−1 in the RB867515 variety, in comparison to the control, respectively. Inoculation and N fertilizer used for the Coruripe plantation increased stem yield of 38.0 and 42.4 Mg ha−1, respectively, with the RB867515 variety, while RB72454 showed increases of 16.7 and 37.5 Mg ha−1, both compared to the control. Biological nitrogen fixation was not affected by the treatments, however, both treatments increased the total recoverable sugar yield. Benefits from inoculation appeared to promote plant growth due to the plant–bacteria interaction.

Benefits of inoculation, P fertilizer and manure on yields of common bean and soybean also increase yield of subsequent maize

Common bean and soybean yield poorly on smallholder farms in Rwanda. We evaluated the benefits of inoculation combined with P fertilizer and manure on yields of common bean and soybean in three agro-ecological zones (AEZs), and their residual effects on a subsequent maize crop. In the first season, the treatments included inoculum, three rates of manure, and two rates of P fertilizer, with nine replications (three per AEZ). Both legumes responded well to inoculation if applied together with manure and P fertilizer. Grain yields varied from 1.0tha−1 to 1.7tha−1 in unamended control plots to 4.8tha−1 for common bean and 3.8tha−1 for soybean in inoculated plots with both P and manure addition. The response of common bean and soybean to inputs varied greatly between AEZs. In the AEZ with low and erratic rainfall (Bugesera), yields of both legumes and maize were low and maize after soybean failed to yield any grain due to drought. In this regard, early maturing legume varieties are advised in regions of low rainfall. Responses of maize to an input applied to the legumes strongly increased when other inputs were applied together to the legume. This allowed greater maize yields which ranged from 0.8tha−1 in control plots to 6.5tha−1 in treatments previously inoculated with P and manure added for maize grown after common bean and from 1.9tha−1 in control plots to 5.3tha−1 for maize grown after soybean. The amount of N2-fixed measured using the 15N-natural abundance method differed between the two legumes and varied between 15 and 198kgN2ha−1 for common bean and between 15 and 186kgN2ha−1 for soybean and differed enormously among treatments and AEZs. Application of inputs to the legumes also resulted in enhanced N and P uptake of the subsequent maize. The use of inoculum combined with manure and P fertilizer is a good option for smallholder farmers growing common bean and soybean in rotation with maize. We observed strong effects of environment and call for care when targeting crops and technologies for sustainable crop production.

Split-root labelling to investigate 15N rhizodeposition by Pinus sylvestris and Picea abies

ABSTRACTWe investigated the transfer of 15N into the soil via 15N uptake and release by tree roots, which involves the principles of the split-root technique. One half of the root system received an injection of (15NH4)2SO4 and the other half equivalent amounts of (NH4)2SO4 at 15N natural abundance level. 15N was transferred from one side of the root system (15N side) to the other side (14N side) and released into the soil. The method was conducted with Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies [L.] Karst). Two concentration levels of (NH4)2SO4 were used, corresponding with annual N deposition in the Netherlands (30 kg N ha–1) and a twelfth of that (2.5 kg N ha−1). Samples were taken 3 and 6 weeks after labelling and divided into needles + stem, roots, rhizosphere and bulk soil. Already 3 weeks after labelling, Scots pine took up 23.7 % of the low and 9.1 % of the high amounts of 15N, while Norway spruce took up 21.5 and 32.1 %, respectively. Both species transported proportions of 15N to the rhizosphere (0.1–0.2 %) and bulk soil (0.3–0.9 %). The method is a useful tool to investigate the fate of root-derived N in soils, for example, for the formation of stable forms of soil organic matter.

Growth patterns of common bean cultivars affect the ‘B’ value required to quantify biological N2 fixation using the 15N natural abundance technique

The ‘B’ value, the 15N abundance of plants depending completely on biological nitrogen fixation (BNF) for growth, is required for quantifying BNF using the 15N natural abundance technique. This study aimed to investigate how common bean (Phaseolus vulgaris L.) cultivars with different growth patterns could affect the ‘B’ value calculated for the plant shoot. Two experiments were conducted in N-free solution. Experiment I had a factorial design with three cultivars and two rhizobium inoculants. Plants were harvested at pre-flowering stage. Experiment II was also factorial with two cultivars and two times of sampling: full flowering and mid-pod filling. Total N and δ15N analyses of different plant parts were carried out to estimate the ‘B’ value. There were differences between cultivars for N accumulation and 15N abundance, but no difference between rhizobium inoculants. Results revealed differences in ‘B’ value between cultivars of small and large seeds, the growth stage being also relevant. The ‘B’ values of −2.08 and −1.34‰ could be used for BNF estimates in small-seeded cultivars at full flowering and mid-pod filling stages, respectively, while such values should change, respectively, to −1.73 and −1.03‰ for large-seed cultivars. The mean ‘B’ value in shoots was −1.90 and −1.20‰ at full flowering and mid-pod filling stages, respectively. There are variations in ‘B’ value between cultivars of different cycle length and seed sizes, which are associated with plant architecture, thus a ‘B’ for each growth stage of common bean plants is required.

Inter-species and intra-annual variations of moss nitrogen utilization: Implications for nitrogen deposition assessment

Moss nitrogen (N) concentrations and natural 15N abundance (δ15N values) have been widely employed to evaluate annual levels and major sources of atmospheric N deposition. However, different moss species and one-off sampling were often used among extant studies, it remains unclear whether moss N parameters differ with species and different samplings, which prevented more accurate assessment of N deposition via moss survey. Here concentrations, isotopic ratios of bulk carbon (C) and bulk N in natural epilithic mosses (Bryum argenteum, Eurohypnum leptothallum, Haplocladium microphyllum and Hypnum plumaeforme) were measured monthly from August 2006 to August 2007 at Guiyang, SW China. The H. plumaeforme had significantly (P < 0.05) lower bulk N concentrations and higher δ13C values than other species. Moss N concentrations were significantly (P < 0.05) lower in warmer months than in cooler months, while moss δ13C values exhibited an opposite pattern. The variance component analyses showed that different species contributed more variations of moss N concentrations and δ13C values than different samplings. Differently, δ15N values did not differ significantly between moss species, and its variance mainly reflected variations of assimilated N sources, with ammonium as the dominant contributor. These results unambiguously reveal the influence of inter-species and intra-annual variations of moss N utilization on N deposition assessment.

Legume inoculant application methods: effects on nodulation patterns, nitrogen fixation, crop growth and yield in narrow-leaf lupin and faba bean

Liquid and granular rhizobial inoculants have some practical advantages for delivering rhizobial inoculants to legume crops in terms of ease-of-use and in separating rhizobia from potentially harmful seed-applied pesticides. The aim of this research was to determine whether inoculant application methodologies altered the patterns of nodulation on roots, inputs of symbiotic nitrogen (N2) fixation, the accumulation of legume shoot dry matter (DM), grain yield, and grain nitrogen (N). Eight field experiments were established at four different locations in south-eastern Australia to quantify the response of lupin (Lupinus angustifolius L.) and faba bean (Vicia faba L.) to three inoculant application methods (on-seed application as a peat slurry, in-furrow peat inoculant delivered as a liquid suspension at seeding, in-furrow peat granules delivered at seeding) compared with uninoculated treatments. N2 fixation was assessed using the 15N natural abundance method and canola was included as a non-legume reference. Inoculation significantly improved crown nodulation, from 0.05 to 13 nodules plant−1 in lupin at two sites and from 0.17 to 21.3 nodules plant−1 in faba bean at three sites. Nodulation responses were decreased for faba bean treatments at sites with low pH, and for both lupin and faba bean at sites where soils contained large populations of naturally-occurring rhizobia. Inoculation increased grain yield from 0.48 to 1.94 t ha−1 in faba bean relative to uninoculated treatments at two sites; N2 fixation increased by 175 kg N ha−1 in lupin at one site and by 46 to 280 kg N ha−1 in faba bean at two sites. The different inoculant application methods led to minor differences in crown and lateral root nodulation patterns but only impacted N2 fixation and grain yield at one site with faba bean, where peat slurry treatments had 186 to 195 kg N ha−1 more N2 fixation than other treatments and peat slurry and granules provided 0.8 to 1.0 t ha−1 more grain yield than liquid inoculants. On-seed application of peat slurry always provided the best nodulation, grain yield and N2 fixation. Small changes in nodulation patterns using in-furrow inoculants only resulted in reduced N2 fixation in faba bean at one site. At that site faba bean grain yield was reduced by 1.0 t ha−1 in liquid inoculant treatments, compared with on-seed peat slurry treatments.

The effect of roots and easily available carbon on the decomposition of soil organic matter fractions in boreal forest soil

The priming effect induced by carbon (C) that is easily available to microbes has been shown to increase the mineralization of soil organic matter (SOM) that is resistant to decomposition, but the combined effects of easily available carbon and the living root system have rarely been studied. The aim of this research was to study the decomposition of SOM fractions of different solubility in water and their 13C, 14C and 15N abundance with and without the presence of a living root system and easily available carbohydrate in the form of glucose. The SOM collected from the organic horizon of a boreal forest soil in Hyytiälä, southern Finland (61°51′N, 24°17′E), and exposed to laboratory incubations with and without the presence of Pinus sylvestris L. seedlings and glucose, was separated into three chemical fractions with accelerated solvent (ASE) and pressurized hot water extractions (PHWE). Changes in the natural abundance of 13C, 14C and 15N, spectral properties assessed by Fourier transform infrared spectroscopy (FTIR) and the C and N pools of SOM fractions were studied after incubation for 6 months. The extractions separated SOM into fractions with distinctive isotopic composition. The most easily soluble SOM fraction showed the largest abundance of 15N and 14C, and the living root system induced changes in the abundance of 15N and FTIR spectra. Our research suggests that plant roots may induce SOM degradation and N uptake from soluble SOM fractions, but 13C, 14C, 15N or FTIR alone cannot be used to describe the recalcitrance of SOM and its accessibility to microorganisms. It is better to use several methods in parallel to study the decomposability of SOM.HighlightsWe studied the effects of tree roots and addition of glucose on the solubility of soil organic matter (SOM). SOM can be separated into pools with distinctive isotopic concentrations with pressurized hot water. Roots may induce organic matter degradation and N uptake from soluble SOM fractions. Larger 14C abundance in soluble SOM indicated it contained older C than the recently assimilated C.

Elevated Atmospheric Carbon Dioxide and Temperature Affect Seed Composition, Mineral Nutrition, and 15N and 13C Dynamics in Soybean Genotypes under Controlled Environments

The seed nutrition of crops is affected by global climate changes due to elevated CO2 and temperatures. Information on the effects of elevated CO2 and temperature on seed nutrition is very limited in spite of its importance in seed quality and food security. Therefore, the objective of this study was to evaluate the effects of elevated atmospheric CO2 and temperature on seed composition (protein, oil, fatty acids, and sugars) and mineral nutrition in two soybean cultivars under controlled environments. The treatments were ambient CO2 concentrations (360 μmol mol-1) and elevated CO2 concentration (700 μmol mol-1) as well as normal temperature (26/16°C) and elevated temperature (45/35°C). Plants were grown under greenhouse conditions until the R5 stage, and then, transferred to growth chambers until full maturity (R8). Elevated temperature or a combination of elevated temperature and elevated CO2 resulted in a decrease in seed protein and linolenic acid concentrations and an increase in oil and oleic acid in cultivars Williams 82 (MGIII) and Hutcheson (MG V). Seed sucrose, glucose, and fructose decreased, whereas raffinose and stachyose remained relatively stable. Minerals also decreased under elevated CO2 and temperature. Among those that decreased were N, P, K, Zn, Fe, and B. Natural abundance of 15N and 13C isotopes was altered only under high temperature, regardless of CO2 concentration, indicating that changes in nitrogen and carbon metabolism occurred at elevated temperature. The increase in oil and oleic acid and decrease in linolenic acid are desirable, as high oleic acid and low linolenic acid contribute to the stability and longer shelf-life of oil. The combination of low protein and high oil was due to the inverse relationship between them. This study showed that seed composition and seed mineral nutrients can be affected by elevated temperature alone or elevated CO2 and temperature. This information is beneficial for selecting varieties with high seed nutritional qualities and efficient mineral nutrient use and uptake, traits that are related to seed production, seed quality, and food security. Also, it provides further knowledge on the effect of climate change on seed quality.

Effect of pyraclostrobin on disease control, leaf physiology, seed yield and quality of sunflower

A two-year (2012–2013) field experiment was carried out to investigate the fungicidal activity of pyraclostrobin and its putative physiological effects on sunflower (Helianthus annuus L.) grown under rainfed, Mediterranean conditions. Five pyraclostrobin treatments were applied [single applications at rates of 100, 150 and 200 g active ingredient (ai) ha−1 at V6 growth stage (TV6/100, TV6/150, and TV6/200, respectively) and 200 g ai ha−1 at R1 growth stage (TR1/200) and a double application at V6 and R1 at a rate of 100 g ai ha−1 (TV6-R1/100)] and accompanied by untreated (TUC) and chemical control [iprodione at a rate of 750 g ai ha−1 at V6 (TCC)]. Diseases [charcoal rot (CR) caused by Macrophomina phaseolina, Phomopsis stem canker (SC) caused by Phomopsis helianthi, black stem (BS) caused by Phoma macdonaldii, and powdery mildew (PM) caused by Golovinomyces cichoracearum] were assessed twice. Leaf physiological traits [leaf chlorophyll content (SPAD), specific leaf area (SLA), carbon isotope discrimination (Δ), nitrogen (N) concentration, 15N natural abundance (δ15N), and carbon to nitrogen ratio (C/N)] were determined at R5 stage. Growth season had a significant effect on seed oil concentration (Oil) and oil yield (OY) and was higher in 2013; this season had low CR and SC incidence but high incidence of BS and severity of PM. Split application of pyraclostrobin (TV6-R1/100) or a high rate at the beginning of the reproductive phase (TR1/200) suppressed the diseases, mainly SC and BS. The former treatment (TV6-R1/100) showed the highest yields [seed yield (SY), OY, and protein yield (PY)]. All the physiological traits but Δ were not affected by fungicide treatments. The high-yielding TV6-R1/100 treatment had the highest Δ values over the growth seasons indicating a “waste” of water in turn to sustain open stomata and thus higher CO2 exchange rates. This swap leads to lower water use efficiency in the long-term but it indicates healthier and more active functioning sunflower plants that had greater yields. Phomopsis stem canker (SC) showed to be the most detrimental disease since its incidence was negatively correlated to yields (SY, OY) over the growth seasons. On the other hand, CR seemed to affect N uptake and translocation within sunflower plants. In conclusion, the physiological response (highest Δ) recorded for split application of pyraclostrobin (at V6 and R1 stages) was indirect; lower disease incidence secured water supply to sunflower plants resulting in higher yields.

Nitrogen input &amp;lt;sup&amp;gt;15&amp;lt;/sup&amp;gt;N signatures are reflected in plant &amp;lt;sup&amp;gt;15&amp;lt;/sup&amp;gt;N natural abundances in subtropical forests in China

Abstract. Natural abundance of 15N (δ15N) in plants and soils can provide time-integrated information related to nitrogen (N) cycling within ecosystems, but it has not been well tested in warm and humid subtropical forests. In this study, we used ecosystem δ15N to assess effects of increased N deposition on N cycling in an old-growth broad-leaved forest and a secondary pine forest in a high-N-deposition area in southern China. We measured δ15N of inorganic N in input and output fluxes under ambient N deposition, and we measured N concentration (%N) and δ15N of major ecosystem compartments under ambient deposition and after decadal N addition at 50 kg N ha−1yr−1, which has a δ15N of −0.7 ‰. Our results showed that the total inorganic N in deposition was 15N-depleted (−10 ‰) mainly due to high input of strongly 15N-depleted NH4+-N. Plant leaves in both forests were also 15N-depleted (−4 to −6 ‰). The broad-leaved forest had higher plant and soil %N and was more 15N-enriched in most ecosystem compartments relative to the pine forest. Nitrogen addition did not significantly affect %N in the broad-leaved forest, indicating that the ecosystem pools are already N-rich. However, %N was marginally increased in pine leaves and significantly increased in understory vegetation in the pine forest. Soil δ15N was not changed significantly by the N addition in either forest. However, the N addition significantly increased the δ15N of plants toward the 15N signature of the added N, indicating incorporation of added N into plants. Thus, plant δ15N was more sensitive to ecosystem N input manipulation than %N in these subtropical forests. We interpret the depleted δ15N of plants as an imprint from the high and 15N-depleted N deposition that may dominate the effects of fractionation that are observed in most warm and humid forests. Fractionation during the steps of N cycling could explain the difference between negative δ15N in plants and positive δ15N in soils, and the increase in soil δ15N with depths. Nevertheless, interpretation of ecosystem δ15N from high-N-deposition regions needs to include data on the deposition 15N signal.

English

The global nitrogen (N) cycle is markedly, and increasingly, influenced by anthropogenic inputs. A large unknown remains the quantity of biological N fixation (BNF) inputs derived from agriculture. This leads to major uncertainties in modeling reactive N interactions with climate change, and understanding N biogeochemical processes. Understanding N dynamics is central to enhancing productivity in cropping systems. To fill this gap, we used 15N natural abundance to quantify BNF and yield of groundnut and pigeonpea – on 18 on-farm sites in Ekwendeni, Northern Malawi. The study was conducted over the 2007/08 (2008) and 2008/09 (2009) cropping seasons under farmer management, for a range of edaphic environments. Overall, the soils are largely sandy with low to moderate organic carbon (0.12-1.56%), pH (5.5-6.5), and very low to moderately high inorganic P (3 to 85 mg kg-1). Intercropping was efficient at utilization of growth resources than sole cropping as evidences by land equivalent ration (LER) >1. The main drivers of BNF were plant density, inorganic P and interspecific competition. The proportion of N derived from the atmosphere (22-99%) was influenced by soil P status across seasons and crop species, but not by cropping system. The mean proportion of BNF was high in both groundnut (75%) and pigeonpea (76%). Total N fixed, on the other hand, differed with cropping system in the dry year, where intercropping was associated with low levels of N fixed by pigeonpea (15 kg N ha-1) compared to sole pigeonpea (32 kg N ha-1). A short rainfall season could not support biomass production of pigeon pea, and this has negative implications for relying on BNF to drive productivity on smallholder farms. Key words: Intercropping, Groundnut, Pigeonpea, Nitrogen fixation, 15N natural abundance.

15N methodologies for estimating the transfer of N from legumes to non-legumes in crop sequences

A suite of 15N-based methodologies have been applied to quantify the transfer of symbiotically-fixed or legume N to succeeding non-legumes in crop sequences. The structure of these methods, their comparative advantages and efficacy are scrutinized in the present review. Methods are either direct or indirect, the former involving labelling of the legume with 15N2, the substrate for symbiotic N2 fixation, while in the indirect methods the legume is either enriched or depleted in 15N through addition of labelled fertilizer or is otherwise unamended at 15N natural abundance. The methods can be classified further according to their yield dependency, and whether they involve the relocation of residues or are located in situ as green manures or harvest residues. The greatest number of studies has involved the relocation of 15N-labelled plant materials produced artificially at locations remote from the actual crop sequence in the field. Depending on the physico-chemical properties of the residues and the environmental and edaphic conditions pertaining at the experimental site, the proportions of non-legume N in phase 2 of the rotation derived from the phase 1 legume have been quite variable. Nevertheless, there are many recorded instances where green manures and harvest residues represent significant sources of N in legume–non-legume crop sequences. The application of 15N-based methods has resulted in a better understanding of the relative contribution of below- and above-ground legume N in N transfer, and the efficacy of management practices such as incorporation of green manures or use of surface mulches.

Modified Method for Trapping and Analyzing 15N in NO Released from Soils.

15N isotope tracing is an effective and direct approach to investigate sources of nitric oxide (NO) formed in soils. However, NO is highly reactive and rapidly converted to nitrogen dioxide (NO2) in the presence of ozone, making it impossible to directly measure 15N in NO. Various wet-chemical methods for conversion of NO to nitrite (NO2-) and nitrate (NO3-) have been proposed for 15N analysis in high-concentration NO sources, such as combustion processes. In contrast, NO concentrations in the soil surface-near air are usually small (ppbv-range), posing major challenges to conversion efficiency and blank correction. Here, we present a modified method in which NO is oxidized quantitatively to NO2 by chromium trioxide (CrO3), before conversion to NO2- and NO3- in an alkaline hydrogen peroxide (H2O2) solution. A denitrifier method was used to reduce NO2- and NO3- in the trapping solution quantitatively to nitrous oxide (N2O) for subsequent 15N analysis. NO trapping efficiencies of >85% were obtained with 50 ppb NO in a 0.5 L min-1 air stream bubbling through a solution of 1.2 M H2O2 and 0.5 M NaOH. In a laboratory test with distinct 15NO abundances, the overall precision was 0.29‰ (δ-values) for natural abundance NO and 0.13 atom % for labeled NO, suggesting that our method can be used for both natural abundance studies and 15N labeling experiments. In a soil incubation experiment with 15NH4NO3, NH415NO3, or Na15NO2 amendments, we found distinct 15N abundances in NO, indicating that our method is well suited to investigate NO sources in soils.