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Damoglou, and Cooper, 1985; Nelson, 2004) on account of its highly efficient, cost-free capture of the nutrient as opposed to using chemical fertiliser inputs and the absence of direct nitrogen leakage to the environment. However, delivering the 'best fit' local Rhizobium as biofertilisers for local clover varieties is a major challenge given the complexity of the host (clover), its selection of Rhizobium for effective symbiosis in a dynamic, harsh soil environment. Nevertheless Rhizobium as clover seed inoculants is desirable, and has not been fully explored for its potential in Ireland’s crop management strategies. The prospects of developing seed inoculants may vastly improve by taping into the existing knowledge accumulated upon seed versus effective rhizobia compatibility mechanisms. Taking cue from the findings that effective partnership for the successful symbioses is encrypted in the ‘early dialogue’ i.e. ‘early nodulin’ (Enod) protein encoding BNF gene expression (Cooper, 2007), in this study, we set out to capture snap shots of early nodulin (Enod) finger-prints of clover roots during early stages of bacterial infection and to explore its usefulness as a gene marker in clover seed-inoculant Rhizobium choices.
2. Materials & Methods White clover (Trifolium repens) seedling roots were challenged with strains of Rhizobium leguminosarum bv trifolii with varying nitrogen-fixing efficiency. The putatively expressed proteins were initially separated on a gel electrophoresis from root hairs and were further purified via an anion exchange column perfusion chromatography (BioCad Sprint) system. We obtained snap shot proteomic data upon these presumptive early nodulins via MALDI-Tof mass spectra analyses and the protein sequences subjected to bioinformatic analyses in tandem with the available genomic data (reverse genetic expression analyses and micro-array genomic databases) comprising Trifolium repens-Rhizobium symbiosis.
55.14 74.92 It was evident from the experimental results that the type of Rhizobium (effective, ineffective or infective but inefficient N-fixer) was recognized by the host plant in terms of the nodulin protein expressed. The clover root early nodulin (Enod) expressed by effective Rhizobium (lane 2-5) distinctly favors the most effective bacterium for symbioses whilst the Enod expressions are either feint for either ineffective Rhizobium (lane 6) or absent altogether in infective but poor N-fixer strains (lane 7, 8). The application of MALDI-Tof in our analyses has highlighted the importance of protein sequence’s coherence of this early dialogue Enod gene expression. Upon closer dissection of the protein sequences, it revealed that a common signal peptide is shared by all full-length Enods, as is a highly conserved Proline-rich region.
The high percentage of Alanine, Glycine, Proline and Serine residues, (see ion peak attenuations in Figure
2) found in the central backbone structure of Enods is characteristic of ‘arabinogalactan’ proteins.
It is perceivable from our proteomic study that the type of Rhizobium (effective, ineffective or infective but inefficient N-fixer) recognized by the host plant and the Enod protein expressed concurs with earlier extensive molecular genetic expression analyses at AFBINI (e.g. Crockard et al., 2001; 2002). The application of MALDI-Tof technique further corroborates the significance of the specificity seen in amino acid positions whose complementary Enod genetic codes were identified amongst the early cognizant signals between clover root and the compatible Rhizobium. Our results suggest that the characteristic ‘arabinogalactan’ responsive proteins in white clover challenged by ‘effective’ rhizobia could serve as a ‘biomarker’ for ensuring ‘efficient’ dialogue taking place between the plant and the bacteria for enhanced biological nitrogen fixation management.
Conclusion Our study showed that the precise ‘early nodulin protein motif’ described herein can be considered as a biomarker for effective host (clover) selection of the type of Rhizobium it symbioses with; thus raising realistic prospects of utilizing this as a useful gene marker in clover seed-inoculant rhizobia choices. Our work also suggests that this protocol can be adaptable for selecting best performing free-living N-fixers (e.g. Azospirillum, Azotobacter) for grass. In the light of our findings, we recommend a twin-track strategy comprising clover and grass seed microbial inoculants technology development for suppressing chemical fertiliser dependency and for improving long-term N-management in Irish grassland swards.
References Anon. 2007. Balanced fertilisation to minimise total Nitrogen input with fertiliser (both bio manure and chemical fertiliser) to crop requirements. In EC Nitrates Directive (http://ec.europa.eu/environment/water/waternitrates/index_en.html) Crockard, M., Bjourson, A.J., Pulvirenti, M.G. and Cooper, J.E. 2001. Temporal and spatial expression analyses of TrEnod40, TrEnod5 and a novel early nodulin in white clover roots and nodules. Plant Science 161(6):1161-1170.
Crockard, M., Bjourson, A.J., Dazzo, F.B. and Cooper, J.E. 2002. A white clover nodulin gene, dd23b, encoding a cysteine cluster protein, is expressed in roots during the very early stages of interaction with Rhizobium leguminosarum biovar trifolii and after treatment with chitolipooligosaccharide Nod factors. Journal of Plant Research 115(6):439-447.
Cooper, J. E. 2007. Early interactions between legumes and rhizobia: disclosing complexity in a molecular dialogue. J Appl Microbiology 103 (5), 1355-1365.
Damoglou, A. P. and Cooper, J. E. 1985. Microbial inoculants for legumes and silage - a case to be proved. Agriculture in Northern Ireland 59(12), 401-403.
Nelson, L. M. 2004. Plant growth promoting rhizobacteria (PGPR): Prospects for new inoculants. Online. Crop Management.
Nitrogen Workshop 2012
Fertigation techniques to increase the nitrogen use efficiency of slurries Mantovi, P.a, Bortolazzo, E.b, Battilani, A.c a CRPA Foundation Studies and Researches, Reggio Emilia, Italy b Research Centre on Animal Production – CRPA, Reggio Emilia, Italy c Consorzio di bonifica per il Canale Emiliano Romagnolo CER, Bologna, Italy
1. Background & Objectives The clarified fraction of livestock slurries can be mixed with irrigation water to fertigate crops. New applications are being developed in different continents (Sun et al., 2011).
The solid/liquid separation of slurry concentrates ammonia nitrogen in the clarified fraction while most of the organic matter is removed as solid fraction. By using the clarified fraction, manure nitrogen use efficiency (NUE) is significantly improved compared to unprocessed slurry.
With the aim of improving the NUE of livestock slurries, trials were set up to distribute clarified fractions through fertigation systems on (i) drip fertigated maize (clarified fractions of digested pig slurry and digested cattle slurry), and on (ii) grassland fertigated using a reel machine equipped with boom (clarified fraction of digested cattle slurry).
2. Materials & Methods The supernatant of anaerobically digested pig slurry, following settlement in a storage tank, was diluted with water at a 1:3 ratio (vol/vol) before injection into drip lines. The clarified fraction of anaerobically digested cattle slurry obtained from drum press separation treatment was applied through two different systems, a drip line and a sprinkler. Because of the higher suspended solids content, the cattle slurry clarified fraction needed to be diluted at a 1:10 ratio to be used with drip lines. The diluted clarified fractions were filtered before being injected into the drip irrigation system. Filtration intensity was optimised in order to reduce its cost; therefore a disc filter unit was utilised with pig slurry and a sand filter with cattle slurry. The physical and chemical characteristics of the clarified slurries utilised in fertigation, before and after dilution, are shown in table 1.
Traditional broadcast mineral fertilisation and organic fertilisation with untreated slurries distributed at the earlier maize growing stages have been compared with fertigation. In all treatments, drip lines were positioned between alternate maize rows.
Grassland was sprinkler irrigated and fertigated by a reel machine+boom. Sprinkler irrigation, even when small nozzles are used, is far less affected by clogging caused by the residual suspended solids of the cattle liquid fraction (than drip lines). Therefore, the clarified fraction of cattle slurry was directly injected into the fertigation system (diluted with water at a 1:10 ratio) without further filtration. Three grasslands in different forage crops rotations were fertigated.
To determine nitrate concentration, soil samples were taken to 50 cm depth, close to the drip lines
With respect to ammonia emission, drip fertigation resulted in significant benefits with a reduction of losses of more than 90% in case of pig slurry compared to the band spreading application.
In the fertigated trials nitrate concentration in soils increased after each fertigation and decreased afterwards because of crop uptake. To limit nitrogen leaching as much as possible and to improve the NUE it is of the utmost importance to decide fertigation timing and N supply on the basis of a water and nitrogen balance. Nitrogen application should not exceed plant need and should be performed when the soil water is not moving out of the rootzone. The Maximum Application Standards (MAS) rules, as defined in the Italian Action Programmes under the Nitrates Directive, must be fulfilled.
4. Conclusion Different fertigation techniques offer the possibility to maximise slurry NUE. Therefore, they could help in improving the utilisation of manure nitrogen as a resource and reduce mineral N inputs. The drawbacks in the operational implementation of these systems are the need to set up slurry separation and distribution equipment, and the maintenance of the pumps and filters. Fertigation could be a method to improve and expand the period of manure application during the growing season. Results of these trials will be utilised to scale up the systems at demonstrative level in the LIFE+ AQUA Project coordinated by CRPA (http://aqua.crpa.it).
References Sun Q., Li J., Liu B., Zou G. and Liu B. 2011. Study on biogas slurry drip fertigation technics. China Biogas, 3.
1. Background & Objectives In order to provide farmers with information to help them achieve the requirements of the Northern Ireland Nitrates Action Programme (NI NAP), various farm-management calculators have been developed and deployed over the Internet. This
describes two of these, namely – the Livestock Manure Nitrogen Loading Calculator, and the Crop Nutrient Recommendation Calculator. These calculators were developed jointly by two NI organisations - the Agri-Food & Biosciences Institute (AFBI) and the College of Agriculture, Food and Rural Enterprise (CAFRE). AFBI provided the software development expertise and scientific input, with CAFRE supplying the knowledge transfer with regards EU requirements and the practicality of the calculators. There will be an interactive software demonstration of both online applications.
2. Materials & Methods Livestock Manure Nitrogen Loading Calculator To comply with the NI NAP, farmers need to know if the livestock manure N loading of their farms is below 170 kg N ha-1year-1 limit, or if operating under derogation, whether it is under 250 kg N ha-1year-1 limit. This control is in effect a stocking rate limit which applies to both grazing and intensively farmed livestock. This calculator is an online tool to help farmers to manage their farm businesses to comply with the NI NAP. The calculator uses the following information to estimate farm N loading in addition to simultaneously
calculating the livestock manure phosphorus for farmers operating under derogation:
Area of Land Controlled (per ha): Land owned, leased or let out Livestock details: Numbers and ages of different types of livestock Amounts and types of manure/slurry exported and/or imported Crop Nutrient Recommendation Calculator This calculator estimates the amounts of nitrogen (N), phosphate (P2O5) and potash (K2O) required by crops and thus helps farmers match the nutrient requirements of a crop to that supplied by organic manures and chemical fertilisers. It also assists farmers to comply with the nitrogen and phosphate limits contained within the NI NAP which has a specific requirement that all applications of chemical phosphate fertiliser should be supported by soil analysis and take into account phosphate supplied by livestock manures. In addition the calculator supplies essential information required for NI NAP record keeping for up to five
years. The calculator uses the following information to produce a nutrient management plan:
Soil analysis and previous cropping information Recommendations from both 7th & 8th Editions of RB209; standard figures from NI NAP The farmer inserts information about the amounts and types of slurry and the amounts and types of fertilisers that he intends applying to each field. The calculator uses this information to calculate the total inputs of N, P2O5 and K2O to each field and then shows any differences between these inputs and the amounts actually recommended by RB209. Based on the
differences shown, the farmer may then increase or decrease his manure or fertiliser inputs to obtain a better match.
3. Results & Discussion:
These calculators have proved to be invaluable farm-management tools, helping farmers comply with the NI NAP. Since launch in 2009, the number of unique online users of the Nitrogen Loading Calculator has increased from 418 to 638 in 2011; similarly; the number of users of the Crop Nutrient Recommendation Calculator has risen from 328 in 2009 to 408 in 2011.
4. Conclusion This project has demonstrated successful knowledge transfer, bridging the gap between research and policy with practical application.