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«International 17 Workshop th Nitrogen The was jointly organised by Teagasc and AFBI Printed by Print Depot Suggested citation Authors, 2012. Title ...»

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Acknowledgements We acknowledge the UK’s Defra and Scottish Government in sponsoring Sustainable Arable LINK Project LK09128, and the contributions of ADAS, Agricultural Industries Confederation, Bayer CropScience, British Sugar, Country Land and Business Association, The Co-operative, Frontier, GrowHow, AHDB-HGCA, Hill Court Farm Research, NFU, North Energy Associates, North Wyke Research, PGRO, REA, SAC, Scotch Whisky Research Institute, Soil Essentials, Vivergo fuels, Warburtons and Yara.

References Godfray, H C J, Beddington, J R, Crute, I R, Haddad, L, Lawrence, D, Muir, J F, Pretty, J, Robinson, S, Thomas, S M.

and Toulmin C. 2010. Food Security: The Challenge of Feeding 9 Billion People. Science 327, 812-818.

Rochette, P. and Janzen, H H. 2005. Towards a revised coefficient for estimating N2O emissions from legumes.

Nutrient Cycling in Agroecosystems 73, 171-179.

Nitrogen Workshop 2012

Characterization of indigenous rhizobial strains isolated from faba bean (Vicia faba L.) nodules Blažinkov, M.a, Redžepović, S.a, Stipetić, A.a, Toth, Nb., Sikora, S.a a University of Zagreb Faculty of Agriculture, Department of Microbiology, Zagreb, Croatia b University of Zagreb Faculty of Agriculture, Department of Vegetable Crops

1. Background & Objectives Faba bean is a legume valuable as a significant source of protein rich food and it is used both as a human food and feed for livestok in the Mediterranean region, Middle East, China and Asia. The faba bean contributes to the sustainability of cropping systems via the ability to use atmospheric nitrogen in a symbiotic relationship with nitrogen fixing bacteria Rhizobium leguminosarum bv. viciae, reducing fossil energy consumption and application of mineral nitrogen fertilizers and providing source of nitrogen for future crop (Jensen et al., 2010). The high variations in the amount of fixed nitrogen are a result of genotypic characteristics and symbiotic efficiency of R. leguminosarum bv. viciae strains. The diversity of R.

legumnosarum bv. viciae population has usually been determined by phenotypic and/or genotypic characterisation of strains isolated from legume root nodules (Martinez-Romero, 1994). The main objective of this study was to characterize indigenous R. leguminosarum bv.

viciae strains isolated from faba bean nodules and to determine the genetic and phenotypic diversity in natural populations of faba bean rhizobia.

2. Materials & Methods Nodules were collected from faba bean plants grown in the Mediterranean region of Croatia.

Isolation of strains from surface sterilized nodules was performed following a standard protocol (Vincent, 1970). Twenty one field isolates were obtained from two locations in southern Dalmatia. Two Rh. leguminosarum bv. viciae strains were also included in these investigations as reference (1001) and type (30132) strains. In order to perform phenotypic characterization of indigenous strains, growth characteristics under different temperature conditions, pH values, salt concentration and carbon source assimilation were determined.

PCR amplification of the nodC gene region was used for identification at the species and biovarieties level while RAPD and REP – PCR were used for identification at the strain level.

3. Results & Discussion The existence of indigenous populations of faba bean rhizobia in the area of investigation was determined. The results of phenotypic characterization revealed that, apart from mannitol, all rhizobial strains can utilise a sucrose and lactose as carbon source. Under strong acidic (pH 4.5) and alkalinic (pH 9.5) conditions, none of the strains isolated from the location Korčula could not grow. Strains from the Metković region were more tolerant to acidic conditions and their growth was determined at pH 4.5 and pH 5.0. All indigenous strains could not grow on medium containing more than 0.5% (w/v) NaCl, except strain M7 which could grow in medium containin 1% (w/v) NaCl. All strains tested grew extremely poorly or failed to grow at 37° C and above. After PCR amplification of the nodC gene region, the specific 220 bp product was determined in all strains indicating that indigenous strains belong to the species R. leguminosarum bv. viciae. Analysis of genetic variability among indigenous strains (Figure

1) revealed significant differences between reference strains and all field isolates. Among R.

leguminosarum bv. viciae isolates considerable genetic diversity was also determined. Both RAPD and REP-PCR methods grouped all isolates from location Korčula in one cluster with Nitrogen Workshop 2012 the exception of strain M6 which was grouped with isolates originating from the other location.

–  –  –

Figure 1. Dendrogram of R.

leguminosarum bv.viciae strains derived from a) RAPD fingerprints generated using three different primers and b) REP-PCR fingerprints generated using REP- primers

4. Conclusion The results of the present study revealed considerable genetic diversity among natural rhizobial populations. Further investigations are needed in order to obtain information about symbiotic properties of indigenous faba bean strains.

References Jensen E. S., Peoples M. B. and Hauggaard – Nielsen H. 2010. Faba bean in cropping systems. Field Crop Research 115,203-216.





Köpke, U. and Nemecek, T. 2010. Ecological services of faba bean. Field Crops Research 115, 217-233.

Martinez-Romero, E. 1994. Recent developments in Rhizobium taxonomy. Plant and Soil, 161, 11-20.

Moschetti G., Peluso A., Protopapa A., Anastasio M., Pepe O. and Defez R. 2005. Use of nodulation pattern, stress tolerance, nodC gene amplification, RAPD – PCR and RFLP – 16S rDNA analysis to discrimante genotypes of Rhizobium leguminosarum biovar viciae. Systematic and Applied Microbiology 28, 619-631.

Vincent, J.M. 1970. A manual for practical study of root – nodule bacteria. International Biological Programme Handbook No. 15, London

Nitrogen Workshop 2012

Comparison between grass, leguminous and crucifer species used as cover crops Ramírez-García, J. a, Carrillo, J.M. a, Ruiz M. b, Quemada, M. a* a ETSI Agrónomos, Technical University of Madrid, Ciudad universitaria s/n, 28040 Madrid (Spain) b Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Centro Nacional de Recursos Fitogenéticos, Autovía Aragón Km 36, Apdo. 1045, Alcalá De Henares 28800, Madrid (Spain).

1. Background & Objectives Introducing cover crops in the inter-crop period can entail benefits such as an improvement in weed and erosion control, reduced nitrate leaching, and higher soil nutrient content (Thorup-Kristensen et al., 2003). These crops are used in humid regions with high autumn precipitation, but could have a role in Mediterranean climate if irrigation is available to ensure establishment. Cover crops should use water efficiently, present rapid ground cover and high biomass production. Quality of the cover crop biomass determines both stubble decomposition and nutritional quality as fodder (Quemada and Cabrera, 1995). The objective of this work was to compare the suitability of different species of grass, legume and crucifer to be used as cover crops in semi-arid Mediterranean climate.

2. Materials & Methods A factorial experiment with three replications was conducted in the experimental farm of the

Technical University of Madrid (Central Spain). Factors were cover crops sown in October 2010:

barley (Hordeum vulgare L. cv. Tardana), triticale (x Triticosecale Whim cv. Forricale), rye (Secale cereale L. cv. Petkus), mustard (Sinapis alba L.) and vetch (Vicia sativa L. cv. Aitana). Plot size was

1.2 x 7 m2. Ground cover (GC) was determined by digital image analysis, fraction intercepted of photosynthetic active radiation (FIPAR) by mean of a ceptometer, and aerial biomass by destructive sampling in 17 dates. End of March 2011 cover crops were killed; the C/N of aerial biomass was determined by combustion (Dumas Method) and the dietary fiber content by enzymatic digestion (Van Soest and Robertson, 1991).

3. Results & Discussion Barley reached the highest GC and FIPAR values since early stages, while vetch the lowest (Figure 1). Mustard covered the soil as fast as grasses until January, but after low temperatures lost plant biomass and reduced GC (Figure 2). At the end of the experiment barley and rye covered around 95% of the ground, while mustard 80%. Vetch cultivar selected especially for its resistance to low temperatures reached a 91% of GC. Rye and triticale produced the highest amount of biomass (1800-2100 g m-2), closely followed by the barley, while vetch lowest (750 g m-2). The C/N of the biomass presented a large variation, ranging from 13 for mustard to 35 for barley (Figure 3).

Dietary fiber results revealed the grasses to be the most suitable to use as fodder, based on the high neutro-detergent fiber content and the low lignin (Figure 4). In figures, different letters indicate significant differences at the 0.05 level (Tukey).

4. Conclusions Large differences in the measured variables were observed between cover crops. Grasses and mustard covered the soil faster and provided larger FIPAR in early stages than vetch, but these variables decayed for mustard after temperatures decreased in January. At harvest, vetch reached a high GC with the lowest biomass production. Triticale generated the highest amounts of biomass, but C/N was above 33. Grasses were more suitable for covering the ground and the N content in their biomass was equal to the legume, showing a high capacity to recycle N in the system.

Figure 4. Dietary fiber content: Neutro-detergent fiber (FND%), acid-detergent fiber (ADF%) and lignin (L%) in biomass at the end of the experiment.

Cb: Barley, Cn: Rye, Si: Mustard, Tc: Triticale, Vz: Vetch.

Acknowledgements: Financial support by CICYT (AGL2008-00163/AGR) and CAM (S2009/AGR1630).

References Quemada, M. and Cabrera, M.L. 1995. Carbon and Nitrogen mineralization from leaves and stems of four cover crops. Soil Science Society of American Journal 59, 471-477.

Thorup-Kristensen, K., Magid, J. and Jensen, L.S. 2003 Catch crops and green manures as biological tools in nitrogen management in temperate zones. Advances in Agronomy 79, 227-301.

Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber and non-starch polysaccharides in relation to animal nutrition. Journal of Dairy Science 74, 3583-3597.

Nitrogen Workshop 2012

Consequences of long-term application of alternative N sources on gaseous emissions.

Calleja-Cervantes, M.E.a, Irigoien, N.b, Cibriain, F.c, Lasa, B.a, Aparicio-Tejo, P.a, Menendez, S.a a Instituto de Agrobiotecnología. Public University of Navarre. Navarre. Spain b Departamento de Producción Agraria. Public University of Navarre. Navarre. Spain.

c Navarre’s Station of Viticulture and Oenology. Olite, Navarre. Spain.

1. Background & Objectives Nowadays, alternative nitrogen sources are found in organic wastes that have been composted for their application as soil amendments. Overall, composted organic wastes are substances with desirable agricultural properties; its beneficial effects cover the total of soil functions. However, it is well documented that the increase in greenhouse gases (CO2, CH4 and N2O) concentration in the atmosphere is one of the most detrimental repercussions on the environment due to fertilizer/amendment application. In the midst of understanding N fate with alternative N sources, this work explores if organic amendment application after 14 years has evident consequences on greenhouse gas emission.

2. Materials & Methods To fulfil the abovementioned objectives, this research was carried out in a long-term experiment where a vineyard was established in 1996 in Bargota, Navarre, Spain. The climate is a semiMediterranean, with hot summers and annual rainfalls between 450mm and 490mm. The mean annual temperature is 13.8°C. Soil texture in this area is loamy-clay. The experiment entailed the use of three composted fertilizers (amendments), one mineral and a control treatment which remained unfertilized. The fertilizers are described on Table 1 as follows, a pelletized organic compost of vegetable residues and cattle manure (PEL), a municipal solid waste compost (MSW), an organic compost made of sheep manure (SMC). The mineral (NPK) fertilizer was determined by the annual commercial offer for grapevine. Treatments were replicated following a random experimental design with three blocks; each block contained the five treatments randomly placed.

Organic amendments were applied and incorporated into the soil in February every year for 14 years. In the case of statistical analyses, one-way ANOVA tests were used; to compare differences between treatments, Duncan test were performed with a significance level P 0.05 using the SPSS software (SPSS 17, 2010).

Table 1. Physical and chemical properties of fertilizers.

Pelletized organic compost (PEL), municipal solid waste compost (MSW), sheep manure compost (SMC) and Mineral (NPK)

PEL MSW SMC NPK

–  –  –

N2O, CO2 and CH4 were measured 2,3,5,10,15,21,45,60, 90 and 115 days after fertilization, using closed chambers. Emission rates were determined using closed chamber technique, taking into account the concentration increase with time (Menéndez et al., 2008). Samples were analysed by

Nitrogen Workshop 2012

gas chromatography (Agilent, 7890A) with an electron capture detector. A capillary column (IA KRCIAES 6017: 240ºC, 30 m x 320mm) was used. The column’s temperature ramped from 40ºC to 80ºC and ECD’s temperature was 350ºC, and 5% mixture of Ar, with CH4 was used as carrier and N2 as make up (15 mL min-1). A headspace autosampler (Teledyne Tekmar HT3) was connected to the gas chromatograph. Standards were stored and analysed at the same time as samples.

3. Results & Discussion Although, treatments with organic amendments showed higher cumulative losses than mineral fertilizer, the Duncan test did not show significant differences between treatments (Table 1).

–  –  –



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