Jochen Mayer

Estimating N rhizodeposition of grain legumes using a 15N in situ stem labelling method

Abstract Grain legumes in crop rotations cause significant increases in yield for succeeding non-legumes, which cannot be explained simply by the small effect that legumes have on the soil nitrogen balance, as found in the analysis of N in crop residues. Besides known positive non-N-effects, other effects, mainly rhizodeposition and its contribution to the N balance and nitrogen dynamics after harvesting the grain, are poorly understood. In this study, N rhizodeposition, defined as root-derived N in the soil after removal of visible roots, was measured in faba bean ( Vicia faba L.), pea ( Pisum sativum L.) and white lupin ( Lupinus albus L.). In a pot experiment the legumes were pulse labelled in situ with 15 N urea using a cotton wick method. About 84% of the applied 15 N was recovered for the three legume species at maturity. The 15 N was comparatively uniformly distributed among plant parts. The N rhizodeposition constituted 13% of total plant N for faba bean and pea and 16% for white lupin at maturity, about 80% of below ground plant N, respectively. Some 7% (lupin)–31% (pea) of the total N rhizodeposits were recovered as micro-roots by wet sieving (200xa0μm) the soil after all visible roots had been removed. Only 14–18% of the rhizodeposition N was found in the microbial biomass and a very small amount of 3–7% was found in the mineral N fraction. In pea, 48% and in lupin 72% of N rhizodeposits could not be recovered in the mentioned pools and a major part of the unrecovered N was probably immobilised in microbial residues. The results of this study clearly indicate that N rhizodeposition from grain legumes represent a significant pool for N balance and N dynamics in crop rotations.

Diversity of transcripts of nitrite reductase genes (nirK and nirS) in rhizospheres of grain legumes.

ABSTRACT Transcription of the nirK and nirS genes coding for dissimilatory bacterial nitrite reductases was analyzed by reverse transcription PCR (RT-PCR) of mRNA isolated from rhizosphere samples of three economically important grain legumes at maturity: Vicia faba, Lupinus albus, and Pisum sativum. The nirK gene and transcripts could be detected in all the rhizosphere samples. In contrast, nirS could not be detected. Sampling variations were analyzed by comparing denaturing gradient gel electrophoresis profiles derived from nirK RT-PCR products. High similarity was observed between the replicates, and so one representative product per legume was cloned. Clones with the correct insert size were screened by restriction fragment length polymorphism by using the restriction enzyme MspI. The clones could be distributed into 12 different patterns. Patterns 1, 3, 4, 5, and 7 were common in clone libraries of the three rhizosphere types under study. Patterns 2, 9, 10, and 11 were absent from Pisum rhizospheres, while patterns 6, 8, and 12 were absent from the Vicia library. Pattern 1, which was the most dominant in the Vicia and Lupinus libraries, constituted about 25% of all clones. The Lupinus library had clones representing all 12 patterns, indicating it to be the most diverse among the three. Clones representative of each pattern were sequenced. All patterns grouped together forming a distinct cluster, which was divergent from previously described nirK sequences in the database. The study revealed a hitherto unknown diversity of denitrifiers in legume rhizospheres. A plant-dependent rhizosphere effect on the transcripts of a gene was evident.

Characterization of Bacterial Community Structure in Rhizosphere Soil of Grain Legumes

Molecular techniques were used to characterize bacterial community structure, diversity (16S rDNA), and activity (16S rRNA) in rhizospheres of three grain legumes: faba beans (Vicia faba L., cv. Scirocco), peas (Pisum sativum L., cv. Duel) and white lupin (Lupinus albus L., cv. Amiga). All plants were grown in the same soil under controlled conditions in a greenhouse and sampled after fruiting. Amplified 16S rDNA and rRNA products (using universal bacterial primers) were resolved by denaturing gradient gel electrophoresis (DGGE). Distinct profiles were observed for the three legumes with most of the bands derived from RNA being a subset of those derived from DNA. Comparing the total bacterial profiles with actinomycete-specific ones (using actinomycete-specific primers) highlighted the dominance of this group in the three rhizospheres. 16S PCR and RT-PCR products were cloned to construct libraries and 100 clones from each library were sequenced. Actinomycetes and proteobacteria dominated the clone libraries with differences in the groups of proteobacteria. Absence of β-subdivision members in pea and γ-subdivision members of proteobacteria in faba bean rhizosphere was observed. Plant-dependent rhizosphere effects were evident from significant differences in the bacterial community structure of the legume rhizospheres under study. The study gives a detailed picture of both residing and „active” bacterial community in the three rhizospheres. The high abundance of actinomycetes in the rhizospheres of mature legumes indicates their possible role in soil enrichment after the legumes are plowed into the soil as biofertilizers.

Residual nitrogen contribution from grain legumes to succeeding wheat and rape and related microbial process

The residual N contribution from faba bean (Vicia faba L.), pea (Pisum sativum L.) and white lupin (Lupinus albus L.) to microbial biomass and subsequent wheat (Triticum aestivum L.) and oilseed rape (Brassica napus L.) was studied in a greenhouse experiment. The grain legumes were 15N labelled in situ with a stem feeding method before incorporated into the soil, which enables the determination of N rhizodeposition. Wheat and rape were subsequently grown on the soil containing the grain legume residues (incl. 15N-labelled rhizodeposits) and were harvested either twice at flowering and at maturity or once at maturity, respectively. The average total N uptake of the subsequent crops was influenced by the legume used as precrop and was determined by the residue N input and the N2-fixation capacity of the legume species. The succeeding crops recovered 8.6–12.1% of the residue N at maturity. Similar patterns were found for the microbial biomass, which recovered 8.2–10.6% of the residue N. Wheat and rape recovered about the same amount of residue N. The absolute contribution of soil derived N to the subsequent crops was similar in all treatments and averaged 149 mg N pot−1 at maturity. At flowering 17–23% of the residue derived N was recovered in the subsequent wheat and in the microbial biomass; 70% of the residue N was recovered in the microbial biomass in the flowering stage and decreased to about 50% at maturity. In contrast, the recovery in wheat and rape constituted only 30% at flowering and increased to 50% at maturity in all treatments, indicating that the residual N uptake by the subsequent wheat was apparently supplied by mobilisation of residue N temporarily immobilised in the microbial biomass.

Turnover of grain legume N rhizodeposits and effect of rhizodeposition on the turnover of crop residues

The turnover of N derived from rhizodeposition of faba bean (Vicia faba L.), pea (Pisum sativum L.) and white lupin (Lupinus albus L.) and the effects of the rhizodeposition on the subsequent C and N turnover of its crop residues were investigated in an incubation experiment (168xa0days, 15xa0°C). A sandy loam soil for the experiment was either stored at 6xa0°C or planted with the respective grain legume in pots. Legumes were in situ 15N stem labelled during growth and visible roots were removed at maturity. The remaining plant-derived N in soil was defined as N rhizodeposition. In the experiment the turnover of C and N was compared in soils with and without previous growth of three legumes and with and without incorporation of crop residues. After 168xa0days, 21% (lupin), 26% (faba bean) and 27% (pea) of rhizodeposition N was mineralised in the treatments without crop residues. A smaller amount of 15–17% was present as microbial biomass and between 30 and 55% of mineralised rhizodeposition N was present as microbial residue pool, which consists of microbial exoenzymes, mucous substances and dead microbial biomass. The effect of rhizodeposition on the C and N turnover of crop residues was inconsistent. Rhizodeposition increased the crop residue C mineralisation only in the lupin treatment; a similar pattern was found for microbial C, whereas the microbial N was increased by rhizodeposition in all treatments. The recovery of residual 15N in the microbial and mineral N pool was similar between the treatments containing only labelled crop residues and labelled crop residues + labelled rhizodeposits. This indicates a similar decomposability of both rhizodeposition N and crop residue N and may be attributable to an immobilisation of both N sources (rhizodeposits and crop residues) as microbial residues and a subsequent remineralisation mainly from this pool.

Evaluation of the wick method for in situ 13C and 15N labelling of annual plants using sugar-urea mixtures

To investigate the amount and fate of root-derived C and N, often tracer techniques are used, where plants are labelled with isotopes. In the present study, we evaluated the suitability of the cotton wick method for in situ labelling of peas (Pisum sativum L.) and oats (Avena sativa L.) with 13C and 15N simultaneously. With two greenhouse experiments we investigated how the wick method and aqueous urea and sugar solutions at a variety of concentrations affected plant development. In addition, we investigated the distribution of 13C and 15N in plants from column experiments under outdoor conditions. Solution was taken up by the plant from a small vial connected to the stem by a cotton wick which was passed through a hole in the stem of the plants. Generally, solution uptake varied between individual plants and decreased with increasing sugar concentrations. Below-ground, above-ground and total plant dry matter, were not significantly affected by the wick method and the applied solutions. Mixtures of aqueous glucose solutions at 2 to 4% and aqueous urea solutions at 1% are useful carriers of 13C and 15N. However, in the investigated plants isotopes were not homogeneously distributed among plant parts. Above-ground plant biomass was preferentially enriched with 13C and 15N, whereas below-ground plant biomass was generally lower enriched. Moreover, isotope distribution ratio of individual plants varied considerably, independent of plant part or timing of labelling. This must be taken into account when estimating root-derived C and N. Future studies comparing labelling methods need to present the isotope distribution ratios among plant parts to allow a true comparison of the methods and the evaluation of their suitability for estimating rhizodeposition.

Impact of growing maize (Zea mays) on the decomposition of incorporated fresh alfalfa residues

In this study, the effects of growing maize plants on the microbial decomposition of easily degradable plant residues were investigated in a 90-day pot experiment using a sandy arable soil. Four treatments were carried out: (1) untreated control, (2) with freshly chopped alfalfa residues (Medicago sativa L.) incorporated into soil, (3) with growing maize plants (Zea mays L.), and (4) with growing maize plants and freshly chopped alfalfa residues incorporated into soil. The amount of alfalfa residues was equivalent to 1.5xa0mg Cxa0g−1 soil and 120xa0μgxa0N g−1 soil. At the end of the experiment, only the combination of growing maize plants and alfalfa residues significantly increased the contents of microbial biomass C, microbial biomass N, and ergosterol in soil compared to the non-amended control. The dry weight of the maize shoot material was more than doubled in the treatment with alfalfa residues than without. In treatment (2), 6% of the alfalfa residues could be recovered as plant remains >2xa0mm. In treatment (4), this fraction contained 14.7% alfalfa residues and 85.3% maize root remains, calculated on the basis of δ13C values. This means that 60% more alfalfa-C was recovered than in treatment (2). The reasons for the retardation in the breakdown of alfalfa residues might be water deficiency of soil microorganisms in the increased presence of maize roots. Assuming that the addition of alfalfa residues did not affect the decomposition of native soil organic matter, only 23% of the alfalfa residues were found as CO2 monitored with a portable gas analyzer with a dynamic chamber. The discrepancy is probably due to problems in measuring peak concentrations of CO2 evolution in the two alfalfa treatments at the beginning of the experiment and in the two maize treatments at the end, especially in treatment (4).

Above- and belowground nitrogen distribution of a red clover-perennial ryegrass sward along a soil nutrient availability gradient established by organic and conventional cropping systems

AimsBelowground legume nitrogen (N) composed of roots and rhizodeposition is an important N input to soils, but published data of belowground N vary broadly, probably due to extrapolation from short-term experiments and dissimilar growing conditions. We quantified belowground N inputs of red clover (Trifolium pratense L.) during two consecutive years in a clover-grass sward along a soil nutrient availability gradient.MethodsWe established a red clover-perennial ryegrass (Lolium perenne L.) model sward in microplots located in field plots of the DOK experiment, which has a 33-year history of organic and conventional cropping, resulting in a soil nutrient availability gradient. Four treatments were examined: the zero fertilisation control, bio-organic with half and full dose manure application, and the conventional system with mineral fertilisation at full dose. We studied the development of clover aboveground and belowground N using multiple pulse 15N urea leaf labelling.ResultsBelowground clover N increased over time and with rising nutrient availability and was proportional to aboveground clover N at all times. Belowground clover N amounted to 40% of aboveground clover N during two consecutive years, irrespective of the nutrient availability status. Belowground clover N development was initially dominated by fast root growth, followed by enhanced root turnover during the second year. Potassium availability limited clover growth and total N accumulation in treatments with low nutrient availability.ConclusionsBelowground red clover N inputs could be estimated from aboveground N by a constant factor of 0.4, regardless of the nutrient availability and cultivation time. Root turnover led to a distinct absolute increase of N rhizodeposition over time. Hence, N rhizodeposition, with an 80% share of belowground N, was the predominant N pool at the end of the second year.