Research Article Received: 18 October 2012
Revised: 6 September 2013
Accepted article published: 29 October 2013
Published online in Wiley Online Library: 26 November 2013
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6463
Condensed tannin accumulation and nitrogen fixation potential of Onobrychis viciifolia Scop. grown in a Mediterranean environment Giovanni A Re, Giovanna Piluzza, Leonardo Sulas,∗ Antonello Franca, Claudio Porqueddu, Federico Sanna and Simonetta Bullitta Abstract BACKGROUND: Sainfoin (Onobrychis viciifolia Scop.) is a forage legume found in temperate areas but is less widespread in Mediterranean environments. Compared with other perennial legumes, it has the advantage of containing condensed tannins (CT) that can be important for their implications on ruminant nutrition and health. Data on nitrogen (N) fixation by sainfoin in the literature originate from very different environments and only a few field data are available, so it is important to improve knowledge on the N fixation potential of this species, particularly under a Mediterranean climate. Here the accumulation pattern of polyphenolic compounds (total, non-tannic polyphenols and CT) and the N fixation potential of sainfoin were studied in order to contribute to its valorisation for sustainable farming management in Mediterranean environments. RESULTS: CT concentrations were always in the range considered beneficial for animals, not exceeding 50 g delphinidin equivalent kg−1 dry matter (DM). The regression of aerial fixed N on aerial DM showed a relationship of 22 kg fixed N t−1 aerial DM in a Mediterranean environment. CONCLUSION: A wider exploitation of sainfoin is suggested for production under rain-fed conditions, thus enlarging the limited set of available perennial legumes suitable for Mediterranean environments. c 2013 Society of Chemical Industry Keywords: atmospheric nitrogen; fixed nitrogen; phenological phases; polyphenols; sainfoin
INTRODUCTION
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of condensed tannins (CT) and that, according to the literature, comparisons with other published CT data for similar species are very difficult owing to variations in methods, procedures and standards used for analyses.8 – 11 Analyses of monomeric compounds obtained from CT of sainfoin and sulla (Hedysarum coronarium L.) revealed the presence of delphinidin as the most abundant compound, together with cyanidin, and, according to Tava,12 delphinidin can be used for quantification of cyanidin without any significant error.11 Sainfoin can interact with bacteria from the genera Mesorhizobium, Rhizobium and Bradyrhizobium, can be crossinoculated by Rhizobium species from several other host plant species and can also form arbuscular mycorrhizas.13,14 Data on N fixation by sainfoin available in the literature seem somewhat contradictory in terms of absolute amounts, probably owing to differences in soil fertility; it seems likely that sainfoin is dependent on some mineral nitrogen at early growth stages, regardless of rhizobial identity, and later growth stages benefit significantly from an effective symbiosis.3 Moreover, the N fixation data reported in
∗
Correspondence to: Leonardo Sulas, CNR-ISPAAM, Istituto per il Sistema Produzione Animale in Ambiente Mediterraneo, Traversa La Crucca 3, Localit`a Baldinca, 07100 Sassari, Italy. E-Mail: [email protected] CNR-ISPAAM, Istituto per il Sistema Produzione Animale in Ambiente Mediterraneo, Traversa La Crucca 3, Localit`a Baldinca, 07100 Sassari, Italy
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Sainfoin (Onobrychis viciifolia Scop.) is a perennial temperate tanniferous legume that could be valuable for the improvement of sustainable farming systems because of its feeding value and nitrogen (N) fixation. It is well adapted to dry hilly environments on calcareous soils and is used for grazing, hay making and silage conservation.1,2 It is widespread in temperate zones of North America, Europe and the Middle East.1 According to Hayot Carbonero et al.,3 O. viciifolia has a long history of traditional cropping, but its use has declined in western countries over the last few decades owing to low productivity and persistency, to increased competition from higher-yielding forages (mostly Medicago sativa L. and Trifolium spp.) and, especially in the 1980s, to the Common Agricultural Policy of the European Union that favoured intensive production.3,4 Sainfoin once represented an important forage legume in semiarid environments of Italy, but its cultivation area has decreased from 160,000 to 9,000 ha in the last 30 years.5 This trend is now changing and recently sainfoin has been reappraised for its positive characteristics leading to highly satisfactory animal performance, and it is much appreciated by farmers for its rusticity, ability to restore soil fertility and non-bloating qualities.2,6 Moreover, the species has also been investigated as an energy crop.7 It is important to consider that the use of tanniferous legumes can be one solution for improving protein utilization in ruminants, that protein degradation in the rumen is reduced in the presence
www.soci.org the literature often do not specify the sainfoin variety or the rhizobial identity, and the few field data available originate from very different environments; for these reasons it is interesting to improve knowledge on the N fixation potential of sainfoin, particularly under a Mediterranean climate.15 Considering the expected important benefits from forage legumes in general4 and sainfoin in particular3 and in order to contribute to the valorisation of sainfoin in the frame of sustainable farming in Mediterranean environments under dry conditions, the present study was focused on (1) the accumulation pattern of polyphenolics and (2) the N fixation potential of this leguminous species.
MATERIALS AND METHODS Location and crop management The study was carried out during 2001–2003 at Sassari, NW Sardinia (40◦ 53 N, 8◦ 29 E, 60 m a.s.l.) on a site where, to our knowledge, sainfoin had never been cropped before. The site has a mild Mediterranean climate with a mean annual rainfall of 544 mm, a mean annual air temperature of 16.2 ◦ C and a sandy-loam calcareous soil (Table 1). The total rainfall recorded in 2001–2002 was comparable to long-term values, while during the second year it was about 40% higher. Two stands of sainfoin were established, one for each experiment. Experiment A For the phenolic content determination a stand of sainfoin ecotype ‘Toscano’ was established by sowing viable seed at 60 kg ha−1 . Sulla cv. ‘Grimaldi’ (30 kg ha−1 ) was used to establish a reference species. Plots were arranged in a randomized complete block design with three replicates. The size of each experimental unit was 20 m2 . Plots were fertilized with 100 kg ha−1 P2 O5 using triple superphosphate before seeding. No irrigation, fertilizer or herbicide was applied after sowing. Periodically, at times corresponding to different phenological phases, sainfoin and sulla samples were harvested from the central area of each plot, separated into different plant parts, immediately frozen in liquid nitrogen, freeze-dried and then ground to a fine powder for phenolic content determination.
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establish non-fixing reference species. Plots were arranged in a randomized complete block design with three replicates. The size of each experimental unit was 10 m2 . Plots were fertilized with 100 kg ha−1 P2 O5 using triple superphosphate before seeding. No irrigation, fertilizer or herbicide was applied after sowing. Dry matter (DM) yields for sainfoin and the non-fixing reference species were determined by cutting the aerial biomass at 5 cm above ground level over a 3 m2 area within each experimental unit and drying the cut material at 65 ◦ C in a forced air oven until the obtainment of a constant weight. The composition of the sainfoin biomass was determined by choosing ten plants randomly and separating them into leaves, stems and racemes. At each cutting date, all remaining biomass was cut and removed from each plot to simulate forage utilization. Phenolic content determination (experiment A) Plant tissue samplings for chemical determinations were made at different phenological phases, namely flower bud, flowering, early seed set and regrowth, in accordance with the classification of Borreani et al.16 Sampled plant tissues were separated into different parts, namely leaves, stems and racemes. Total phenolics (TotP) were evaluated using spectrophotometric analysis with Folin–Ciocalteu phenol reagent,11,17 with some modifications. Results were expressed as g gallic acid equivalent (GAE) kg−1 DM. Non-tannic phenolics (NTP) were determined after precipitation of tannin components with polyvinylpolypyrrolidone as reported in FAO/IAEA.18 Results were expressed as g GAE kg−1 DM. The butanol assay of Porter et al.,19 with some modifications, was used for the quantification of extractable CT content.11 Results were expressed as g delphinidin equivalent (DE) kg−1 DM. Quantification of fixed N (experiment B) The quantity of N fixed by sainfoin was assessed by the Isotopic Dilution method.20 The proportion of N derived from the atmosphere (Ndfa ) and the amount of N fixed were calculated from the following expressions: Ndfa (%) = 1 − atom15 N excesssainfoin /atom15 N excessreference plant
Experiment B For the nitrogen fixation experiment a stand of sainfoin ecotype ‘Toscano’ was established at the same sowing rate as in experiment A. Chicory (Cichorium intybus L.) cv. ‘Spadona’ (10 kg ha−1 ) and oats (Avena sativa L.) cv. ‘Perona’ (180 kg ha−1 ) were used to
Table 1. Soil characteristics at experimental site in Sassari, NW Sardinia Characteristic
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Gravel Sand Silt Clay pH(water) Total N P2 O5(available) K2 O(available) Organic C Total limestone
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Value 16% 58% 27% 15% 7.4 1.4 g kg−1 6.5 mg kg−1 109 mg kg−1 12 g kg−1 560 g kg−1
×100
(1)
atom% N excesssample = atom% Nsample −0.3663 atom%15 N in air (2) fixed N kg ha−1 = sainfoin N × (Ndfa /100) (3) 15
15
This method provides an accurate estimate for Ndfa because it compares 15 N enrichment in the sainfoin and in the non-fixing species; it is not directly influenced by the levels of DM or total N yield. A rate of 4 kg N ha−1 of 15 N-enriched fertilizer (10 atom% 15 N-enriched ammonium sulfate) was applied to a 3 m2 area of non-fixing reference species and sainfoin within each experimental unit. The area (3 m2 ) with the enriched 15 N was the same area sampled for the DM yield determination. The biomass samples from these areas were dried to a constant weight in a forced air oven at 65 ◦ C, air equilibrated, weighed and ground finely enough to pass through a 1 mm mesh. The dry biomass material was subjected to elemental analyzer isotope ratio mass spectrometry at the INRA UCBN Unite´ Mixte de Recherche laboratory (Caen,
c 2013 Society of Chemical Industry
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Condensed tannins and nitrogen fixation in sainfoin
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Figure 1. Percentage partitioning of phytomass as dry weight per plant at different phenological phases in sainfoin (sa) and sulla (su).
France) to determine both N content (%) and 15 N (atom%). The results were compared with those already published for fixed N in sulla,21 which were obtained from an analogous experiment carried out at the same location over the same two years using the same methodology. In fact, sulla can be considered the closest perennial forage legume to sainfoin, with identical growth cycle and production pattern. Statistical analysis For each experiment, statistical significance was determined by two-way analysis of variance (ANOVA). Differences between means were assessed by the least significant difference (LSD) test for separation of means between and within species in the experiment on polyphenolic accumulation at different phenological phases and in different plant components. In the N fixation experiment the LSD test was performed for separation of means between species. The significance level was fixed at P ≤ 0.05 for all statistical analyses.
RESULTS AND DISCUSSION Total phenolics and condensed tannins The percentage partitioning of sainfoin and sulla phytomass based on dry weight per plant showed that the contribution of each
component was similar during the examined phenological phases (Fig. 1). Statistically significant differences in average TotP concentration at different phenological phases were found in leaves and stems of sainfoin and in leaves of sulla (Table 2). Comparing the total phenolic concentrations of the two species, no significant differences were observed in leaves and stems; only racemes showed a significant difference, with sulla having a higher concentration in racemes compared with sainfoin. The average NTP concentrations in leaves, stems and racemes of sainfoin were higher than those of sulla and significantly different between phenological phases in leaves and stems of sainfoin and sulla but only in racemes of sulla (Table 3). Statistically significant differences in average CT concentration at all phenological phases were found in all components of sainfoin but only in racemes of sulla (Table 4). Comparing the CT concentrations in leaves of the two species, sulla almost always showed a higher concentration except at flowering, when no statistical difference was seen; the same trend was observed for racemes. The concentration of CT per plant is shown in Fig. 2. For each species the values were always in the range considered beneficial for animals, not exceeding 50 g DE kg−1 DM.22 The average CT concentration in sainfoin was lower than that in sulla. Variations in TotP, NTP and CT contents at different phenological phases and in different plant parts were found by several authors in sainfoin and sulla.5,10,16,23,24 Leaves were the organs with the highest TotP, NTP and CT contents for both sainfoin and sulla, and, according to the phytomass partitioning (Fig. 1), the percentage of leaves was higher at the phenological phase of flower bud in both species, while in the next phases the percentage of leaves decreased, with a marked drop in sulla at early seed set due to the high percentage of stems. The TotP, NTP and CT average concentrations in leaves of sainfoin showed an increase at the flowering phase. According to Frutos et al.9 and Gebrehiwot et al.,10 comparisons with other published CT data for similar species are very difficult owing to variations in the methods, procedures and standards used for analyses. According to Piluzza and Bullitta,11 the Folin–Ciocalteu and butanol/HCl/Fe3+ assays remain the most practical methods for screening large numbers of different plant species for TotP, NTP and CT; it is appropriate to use delphinidin as a standard for CT determination, because this substance was the most abundant constituent from the acid-catalysed cleavage of CT from the tested plant species sainfoin and sulla.11 By the use of this standard, our data are made comparable and underestimation and overestimation are avoided.11,12
Table 2. Average concentration of total phenolics (g GAE kg−1 DM) in sainfoin and sulla at different phenological phases Leaves Phase Flower bud Flowering Early seed set Spring regrowth Autumn regrowth LSD0.05 (within species) LSD0.05 (between species)
Sainfoin
Stems Sulla
40.2 54.5 44.9 48.9 56.5 6.8
40.9 48.7 48.8 46.8 NA 4.3 NS
Sainfoin
Racemes Sulla
19.4 12.9 14.1 13.5 21.8 4.4
17.6 17.5 13.5 14.7 NA NS NS
Sainfoin
Sulla
— 26.9 28.3 30.8 — NS
— 46.8 41.2 50.1 — NS 17.0
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NA, not available; NS, not significant.
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Table 3. Average concentration of non-tannic phenolics (g GAE kg−1 DM) in sainfoin and sulla at different phenological phases Leaves Phase Flower bud Flowering Early seed set Spring regrowth Autumn regrowth LSD0.05 (within species) LSD0.05 (between species)
Sainfoin
Stems Sulla
13.6 16.8 12.7 13.3 15.3 1.5
6.1 9.4 6.5 5.9 NA 0.9
Sainfoin 9.1 7.0 7.1 7.1 5.6 1.0
7.1
Racemes Sulla
Sainfoin
2.3 3.6 2.4 2.7 NA 0.2
Sulla
— 9.4 8.5 9.6 — NS
4.8
— 5.1 4.6 6.8 — 0.8 3.7
NA, not available; NS, not significant.
Table 4. Average concentration of condensed tannins (g DE kg−1 DM) in sainfoin and sulla at different phenological phases Leaves Phase Flower bud Flowering Early seed set Spring regrowth Autumn regrowth LSD0.05 (within species) LSD0.05 (between species)
Sainfoin
Stems Sulla
22.6 34.9 21.3 24.0 28.3 4.4
31.6 37.7 32.9 33.8 NA NS 8.3
Sainfoin
Racemes Sulla
10.0 5.3 8.3 5.7 10.2 2.6
Sainfoin
11.7 13.2 8.2 12.9 NA NS
Sulla
— 15.3 9.3 13.8 — 4.5
4.2
— 19.5 25.6 29.8 — 1.7 12.3
NA, not available; NS, not significant.
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Figure 2. Whole plant average concentration of condensed tannins in sainfoin and sulla at different phenological phases.
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c 2013 Society of Chemical Industry
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Condensed tannins and nitrogen fixation in sainfoin
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Table 5. Aerial DM yield (kg ha−1 ) of sainfoin and non-fixing reference species Date of cutting
Sainfoin
Chicory
Oats
Table 7. Proportion of N derived from atmosphere (Ndfa ) for nonfixing reference species and fixed N in aerial biomass of sainfoin compared with fixed N in aerial biomass of sulla
LSD0.05
Fixed N (kg ha−1 )a
Ndfa (%) Spring 2002 Autumn 2002 Spring 2003 Total
2.9 1.9 3.0 7.8
0.2 3.1 7.8 11.1
7.7 — 5.6 13.1
1.3 0.8 2.6 4.2
The dynamics of CT concentration in growing plants is an important aspect for implications on ruminant nutrition and health. According to the literature, only defined concentrations of forage CT (20–50 g kg−1 DM) can be useful to increase the efficiency of protein digestion and productivity in forage-fed ruminants without depressing rumen fibre digestion or voluntary intake, and to provide cheaper and safer alternatives to the use of synthetic compounds for controlling some diseases under grazing.25 According to Guglielmelli et al.,6 feeding ruminants forage containing CT may also offer potential benefits for depressive effects on rumen methane emissions. Our CT concentrations measured utilizing a delphinidin standard at comparable stages are in the range 23–35 g DE kg−1 DM for sainfoin and in the range 32–38 g DE kg−1 DM for sulla. Taking into account the different method and standard utilized by Guglielmelli et al.6 for CT quantification, it seems anyway that the concentrations we detected might be considered potentially suitable for the reduction of methane emissions by ruminants. Nitrogen fixation potential Aerial DM yield The mean aerial DM yield of sainfoin was higher in the two spring samplings than in the autumn sampling and the contribution of sainfoin to the total biomass represented about 70, 66 and 53% at the three consecutive samplings respectively (Table 5). Sainfoin was not very competitive against weeds. The aerial yields were comparable to those obtained in some other locations in Italy26 – 28 but lower than those obtained at two Italian sites by Borreani et al.16 and Martiniello and Ciola.29 According to Canu,30 a sainfoin crop grown at the same time in a similar environment of Sardinia produced up to 8 t DM ha−1 at the spring cutting, with a contribution of sainfoin to the total DM exceeding 80%. Nitrogen content and 15 N excess of aerial DM The N concentration in sainfoin aerial DM ranged from 2.6 to 4.7% and the level was almost doubled in autumn compared with the spring harvests (Table 6). In fact, in autumn the plant material was mainly represented by leaves, leading to a corresponding crude protein value close to 30%. The 15 N excess of sainfoin aerial DM ranged from 0.0337 to 0.0533 atom% and was significantly lower
Date of cutting Chicory Spring 2002 Autumn 2002 Spring 2003
Oats
LSD
77.7 — 87.9
NS — NS
82.8 83.3 86.7
Sainfoin
Sullab
61.1 ± 4.6 35.6 ± 4.4 74.1 ± 4.8 78.4 ± 7.8 77.4 ± 17.0 151.4 ± 15.0
NS, not significant. a Values are mean ± standard error. b According to Sulas et al.21
than that detected in all samplings of both reference species owing to its dilution with atmospheric N. Nitrogen fixation The two non-fixing reference species generated very similar Ndfa values in spring 2003 and no significant difference was found between them (Table 7). Oats, being an annual reference species, did not allow autumn cutting measurements for comparison. Only chicory was considered further for the quantification of aerial fixed N. The Ndfa achieved by sainfoin, based on chicory as a reference species, ranged from 82.8 to 86.7% and was in agreement with that of other forage legumes such as alfalfa and white clover.31 In spite of the plentiful available data regarding the effects of different strain inoculations in sainfoin and its N fixation under glasshouse conditions estimated by acetylene reduction assays, very little information an Ndfa values of sainfoin obtained in the field by isotopic methods is available.13,14,32 – 34 Provorov and Tikhonovich15 reported an Ndfa value of 80% determined using the isotope dilution method under Russian conditions. In a biodiversity experiment in Germany, Roscher et al.35 reported Ndfa values ranging from about 65 to 75% obtained using the natural abundance method. Sainfoin accumulated 212.6 ± 12.2 kg ha−1 (mean ± standard error) of aerial fixed N during the two-year experiment (Table 7), and the amount of fixed N in autumn, when the N concentration reached about 5% (Table 6), was similar to the spring amounts. To our knowledge, these results represent a first estimate of the potential N fixation of sainfoin obtained in rain-fed Mediterranean conditions of southern Italy. The N fixed by the sainfoin in this experiment was below the usual range of 130–160 kg ha−1 per year reported by Provorov and Tikhonovich15 and quite far from the potential value of 270 kg ha−1 per year proposed by the same authors. Since such data arise from non-Mediterranean areas and neither the sainfoin genotype nor the rhizobial strain was reported, comparisons cannot be considered appropriate. However, the Mediterranean perennial legume sulla accumulated
Table 6. Concentration of N and 15 N excess in aerial biomass of sainfoin and non-fixing reference species 15
N (%) Date of cutting
2.6 4.7 2.9
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Chicory 2.2 2.2 1.8
Oats 1.1 — 0.9
LSD 0.01 0.09 0.07
Sainfoin
Chicory
0.0429 0.0533 0.0337
0.2828 0.3208 0.2994
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Oats 0.1971 — 0.2830
LSD 0.0181 0.0053 0.0298
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Spring 2002 Autumn 2002 Spring 2003
Sainfoin
N excess (atom%)
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Mediterranean environments also by means of trials in multiple locations.
ACKNOWLEDGEMENTS Thanks are due to the technicians Maddalena Sassu, Salvatore Nieddu, Daniele Dettori, Piero Saba and Anton Pietro Stangoni for the technical assistance provided.
REFERENCES
Figure 3. Relationship between fixed N and DM yield in sainfoin.
265 kg ha−1 of aerial fixed N (Table 7), 25% higher than that of sainfoin, at the same location in the same two-year period.21 This difference was mainly due to the difference in DM yield, which reached 12.4 t ha−1 in sulla and was 29% higher than in sainfoin during the same two years. In fact, a strong relationship exists between legume DM yield and fixed N, as the latter is calculated on the basis of DM yield. Nevertheless, to allow full exploitation of the sainfoin crop, its below-ground N, not considered in this experiment, should be taken into account. In fact, the below-ground N includes N of visible roots and N of rhizodeposits from root exudates, sloughedoff root tissue and dead roots and its contribution to the total plant fixed N may even be greater than that present in the legume aerial biomass.36,37 Relationship between fixed N and DM yield The regression of aerial fixed N on aerial DM in sainfoin showed a relationship of 22 kg fixed N t−1 aerial DM (Fig. 3), in agreement with the performances of white clover and other legume pure stands across a broad range of latitudes and about 20% higher than that achieved by a monoculture of sulla at the same location in the same two-year period.21,38 Compared with sulla, this relationship has probably been influenced by the consistent amount of nonlegumes (mainly grasses) present in the sainfoin plots, where they represented about 30–50% of the total DM. In fact, grasses compete strongly for soil N and, in their presence, legumes are forced to rely heavily on N fixation as an N source.39 On the other hand, the same consistent amount of non-legumes negatively affected the performance of sainfoin in terms of fixed N per hectare.
CONCLUSIONS
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Sainfoin field grown under rain-fed Mediterranean conditions showed beneficial CT concentrations that can be considered suitable to contribute to the improvement of animal performance and health. From our results it appears that sainfoin could be comparable to and as efficient as sulla, having equal or higher N fixation potential. The favourable CT contents and the N fixation potential suggest that sainfoin can enlarge the very limited set of perennial leguminous species suitable for the organization of Mediterranean rain-fed forage systems. For these reasons the genetic potential of sainfoin should be better exploited in
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Condensed tannins and nitrogen fixation in sainfoin 20 Warembourg FR, Nitrogen fixation in soil and plant systems, in Nitrogen Isotope Techniques, ed. by Knowles R and Blackburn TH. Academic Press, New York, NY, pp. 127–156 (1993). 21 Sulas L, Seddaiu G, Muresu R and Roggero PP, Nitrogen fixation of sulla under Mediterranean conditions. Agron J 101:1470–1478 (2009). ´ AR, Tannins and ruminant 22 Frutos P, Herv´as G, Gir´aldez FJ and Mantecon nutrition. Spanish J Agric Res 2:191–202 (2004). 23 Molle G, Decandia M, Giovanetti V, Cabiddu A, Fois N and Sitzia M, Responses to condensed tannins of flowering sulla (Hedysarum coronarium L.) grazed by dairy sheep. Part 1: Effects on feeding behaviour, intake, diet digestibility and performance. Livest Sci 123:138–146 (2009). 24 Molle G, Decandia M, Fois N, Ligios S, Cabiddu A and Sitzia M, The performance of Mediterranean dairy sheep given access to sulla (Hedysarum coronarium L.) and annual ryegrass (Lolium rigidum Gaudin) pastures in different time proportions. Small Ruminant Res 49:319–328 (2003). 25 Min BR, Pinchak WE, Anderson RC, Fulford JD and Puchalat R, Effects of condensed tannins supplementation level on weight gain and in vitro and in vivo bloat precursors in steers grazing winter wheat. J Anim Sci 84:2546–2554 (2006). 26 Argenti G, Gullo C, Pardini A, Pazzi G, Piemontese S and Talamucci P, Response of two sainfoin (Onobrychis viciifolia Scop.) varieties to different methods of utilization, in Grassland and Science in Europe, Vol. 1, ed. by Parente G, Frame J and Orsi S. EGF, Zurich, pp. 367–371 (1996). 27 De Falco E, Landi G and Basso F, Production and quality of the sainfoin forage (Onobrychis viciaefolia Scop.) as affected by cutting regime in a hilly area of southern Italy. Cahiers Options M´editerran´eennes 45:275–280 (2000). 28 Pecetti L, Annicchiarico P, Battini F and Cappelli F, Adaptation of forage legume species and cultivars under grazing in two extensive livestock systems in Italy. Eur J Agron 30:199–204 (2009). 29 Martiniello P and Ciola A, The effect of agronomic factors on seed and forage production in perennial legumes sainfoin (Onobrychis
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30
31 32 33 34 35
36 37 38 39
viciifolia Scop.) and French honeysuckle (Hedysarum coronarium L.). Grass Forage Sci 49:121–129 (1994). Canu S, Quantificazione dell’azotofissazione in campo di leguminose foraggere e da granella: risultati di una ricerca biennale condotta nella piana della Nurra [Quantification of nitrogen fixation from field grown forage and grain legumes: results from a two-year experiment carried out in the Nurra plain]. Tesis MgSc, University of Sassari (2003). Hardarson G and Atkins C, Optimizing biological N2 fixation by legumes in farming systems. Plant Soil 252:41–54 (2003). Hume LJ and Withers NJ, Nitrogen fixation in sainfoin (Onobrychis viciifolia) 1. Responses to changes in nitrogen nutrition. NZ J Agric Res 28:325–335 (1985). Hume LJ, Withers NJ and Rhoades DA, Nitrogen fixation in sainfoin (Onobrychis viciifolia) 2. Effectiveness of the nitrogen-fixing system. NZ J Agric Res 28:337–348 (1985). Cash D, Bowman H and Ditterline RL, Montguide: Sainfoin. Montana StateUniversityBulletinMT9321, Montana State University, Bozeman, MT (1993). Roscher C, Thein S, Weigelt A, Temperton VM, Buchmann N and Schulze ND, N2 fixation and performance of 12 legume species in a 6-year grassland biodiversity experiment. Plant Soil 341:333–348 (2011). Fustec J, Lesuffleur F, Mahieu S and Cliquet JB, Nitrogen rhizodeposition of legumes. A review. Agron Sustain Dev 30:57–66 (2010). Wichern F, Eberhardt E, Mayer J, Joergensen RG and Muller T, Nitrogen ¨ rhizodeposition in agricultural crops: methods, estimates and future prospects. Soil Biol Biochem 40:30–48 (2008). Carlsson G and Huss-Danell K, Nitrogen fixation in perennial forage legume in the field. Plant Soil 253:353–372 (2003). Loiseau P, Soussanna JF, Lounault F and Delpy R, Soil N contributes to the oscillations of the white clover content in mixed swards of perennial ryegrass under conditions that simulate grazing over five years. Grass Forage Sci 56:205–217 (2001).
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Revised: 6 September 2013
Accepted article published: 29 October 2013
Published online in Wiley Online Library: 26 November 2013
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6463
Condensed tannin accumulation and nitrogen fixation potential of Onobrychis viciifolia Scop. grown in a Mediterranean environment Giovanni A Re, Giovanna Piluzza, Leonardo Sulas,∗ Antonello Franca, Claudio Porqueddu, Federico Sanna and Simonetta Bullitta Abstract BACKGROUND: Sainfoin (Onobrychis viciifolia Scop.) is a forage legume found in temperate areas but is less widespread in Mediterranean environments. Compared with other perennial legumes, it has the advantage of containing condensed tannins (CT) that can be important for their implications on ruminant nutrition and health. Data on nitrogen (N) fixation by sainfoin in the literature originate from very different environments and only a few field data are available, so it is important to improve knowledge on the N fixation potential of this species, particularly under a Mediterranean climate. Here the accumulation pattern of polyphenolic compounds (total, non-tannic polyphenols and CT) and the N fixation potential of sainfoin were studied in order to contribute to its valorisation for sustainable farming management in Mediterranean environments. RESULTS: CT concentrations were always in the range considered beneficial for animals, not exceeding 50 g delphinidin equivalent kg−1 dry matter (DM). The regression of aerial fixed N on aerial DM showed a relationship of 22 kg fixed N t−1 aerial DM in a Mediterranean environment. CONCLUSION: A wider exploitation of sainfoin is suggested for production under rain-fed conditions, thus enlarging the limited set of available perennial legumes suitable for Mediterranean environments. c 2013 Society of Chemical Industry Keywords: atmospheric nitrogen; fixed nitrogen; phenological phases; polyphenols; sainfoin
INTRODUCTION
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of condensed tannins (CT) and that, according to the literature, comparisons with other published CT data for similar species are very difficult owing to variations in methods, procedures and standards used for analyses.8 – 11 Analyses of monomeric compounds obtained from CT of sainfoin and sulla (Hedysarum coronarium L.) revealed the presence of delphinidin as the most abundant compound, together with cyanidin, and, according to Tava,12 delphinidin can be used for quantification of cyanidin without any significant error.11 Sainfoin can interact with bacteria from the genera Mesorhizobium, Rhizobium and Bradyrhizobium, can be crossinoculated by Rhizobium species from several other host plant species and can also form arbuscular mycorrhizas.13,14 Data on N fixation by sainfoin available in the literature seem somewhat contradictory in terms of absolute amounts, probably owing to differences in soil fertility; it seems likely that sainfoin is dependent on some mineral nitrogen at early growth stages, regardless of rhizobial identity, and later growth stages benefit significantly from an effective symbiosis.3 Moreover, the N fixation data reported in
∗
Correspondence to: Leonardo Sulas, CNR-ISPAAM, Istituto per il Sistema Produzione Animale in Ambiente Mediterraneo, Traversa La Crucca 3, Localit`a Baldinca, 07100 Sassari, Italy. E-Mail: [email protected] CNR-ISPAAM, Istituto per il Sistema Produzione Animale in Ambiente Mediterraneo, Traversa La Crucca 3, Localit`a Baldinca, 07100 Sassari, Italy
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Sainfoin (Onobrychis viciifolia Scop.) is a perennial temperate tanniferous legume that could be valuable for the improvement of sustainable farming systems because of its feeding value and nitrogen (N) fixation. It is well adapted to dry hilly environments on calcareous soils and is used for grazing, hay making and silage conservation.1,2 It is widespread in temperate zones of North America, Europe and the Middle East.1 According to Hayot Carbonero et al.,3 O. viciifolia has a long history of traditional cropping, but its use has declined in western countries over the last few decades owing to low productivity and persistency, to increased competition from higher-yielding forages (mostly Medicago sativa L. and Trifolium spp.) and, especially in the 1980s, to the Common Agricultural Policy of the European Union that favoured intensive production.3,4 Sainfoin once represented an important forage legume in semiarid environments of Italy, but its cultivation area has decreased from 160,000 to 9,000 ha in the last 30 years.5 This trend is now changing and recently sainfoin has been reappraised for its positive characteristics leading to highly satisfactory animal performance, and it is much appreciated by farmers for its rusticity, ability to restore soil fertility and non-bloating qualities.2,6 Moreover, the species has also been investigated as an energy crop.7 It is important to consider that the use of tanniferous legumes can be one solution for improving protein utilization in ruminants, that protein degradation in the rumen is reduced in the presence
www.soci.org the literature often do not specify the sainfoin variety or the rhizobial identity, and the few field data available originate from very different environments; for these reasons it is interesting to improve knowledge on the N fixation potential of sainfoin, particularly under a Mediterranean climate.15 Considering the expected important benefits from forage legumes in general4 and sainfoin in particular3 and in order to contribute to the valorisation of sainfoin in the frame of sustainable farming in Mediterranean environments under dry conditions, the present study was focused on (1) the accumulation pattern of polyphenolics and (2) the N fixation potential of this leguminous species.
MATERIALS AND METHODS Location and crop management The study was carried out during 2001–2003 at Sassari, NW Sardinia (40◦ 53 N, 8◦ 29 E, 60 m a.s.l.) on a site where, to our knowledge, sainfoin had never been cropped before. The site has a mild Mediterranean climate with a mean annual rainfall of 544 mm, a mean annual air temperature of 16.2 ◦ C and a sandy-loam calcareous soil (Table 1). The total rainfall recorded in 2001–2002 was comparable to long-term values, while during the second year it was about 40% higher. Two stands of sainfoin were established, one for each experiment. Experiment A For the phenolic content determination a stand of sainfoin ecotype ‘Toscano’ was established by sowing viable seed at 60 kg ha−1 . Sulla cv. ‘Grimaldi’ (30 kg ha−1 ) was used to establish a reference species. Plots were arranged in a randomized complete block design with three replicates. The size of each experimental unit was 20 m2 . Plots were fertilized with 100 kg ha−1 P2 O5 using triple superphosphate before seeding. No irrigation, fertilizer or herbicide was applied after sowing. Periodically, at times corresponding to different phenological phases, sainfoin and sulla samples were harvested from the central area of each plot, separated into different plant parts, immediately frozen in liquid nitrogen, freeze-dried and then ground to a fine powder for phenolic content determination.
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establish non-fixing reference species. Plots were arranged in a randomized complete block design with three replicates. The size of each experimental unit was 10 m2 . Plots were fertilized with 100 kg ha−1 P2 O5 using triple superphosphate before seeding. No irrigation, fertilizer or herbicide was applied after sowing. Dry matter (DM) yields for sainfoin and the non-fixing reference species were determined by cutting the aerial biomass at 5 cm above ground level over a 3 m2 area within each experimental unit and drying the cut material at 65 ◦ C in a forced air oven until the obtainment of a constant weight. The composition of the sainfoin biomass was determined by choosing ten plants randomly and separating them into leaves, stems and racemes. At each cutting date, all remaining biomass was cut and removed from each plot to simulate forage utilization. Phenolic content determination (experiment A) Plant tissue samplings for chemical determinations were made at different phenological phases, namely flower bud, flowering, early seed set and regrowth, in accordance with the classification of Borreani et al.16 Sampled plant tissues were separated into different parts, namely leaves, stems and racemes. Total phenolics (TotP) were evaluated using spectrophotometric analysis with Folin–Ciocalteu phenol reagent,11,17 with some modifications. Results were expressed as g gallic acid equivalent (GAE) kg−1 DM. Non-tannic phenolics (NTP) were determined after precipitation of tannin components with polyvinylpolypyrrolidone as reported in FAO/IAEA.18 Results were expressed as g GAE kg−1 DM. The butanol assay of Porter et al.,19 with some modifications, was used for the quantification of extractable CT content.11 Results were expressed as g delphinidin equivalent (DE) kg−1 DM. Quantification of fixed N (experiment B) The quantity of N fixed by sainfoin was assessed by the Isotopic Dilution method.20 The proportion of N derived from the atmosphere (Ndfa ) and the amount of N fixed were calculated from the following expressions: Ndfa (%) = 1 − atom15 N excesssainfoin /atom15 N excessreference plant
Experiment B For the nitrogen fixation experiment a stand of sainfoin ecotype ‘Toscano’ was established at the same sowing rate as in experiment A. Chicory (Cichorium intybus L.) cv. ‘Spadona’ (10 kg ha−1 ) and oats (Avena sativa L.) cv. ‘Perona’ (180 kg ha−1 ) were used to
Table 1. Soil characteristics at experimental site in Sassari, NW Sardinia Characteristic
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Gravel Sand Silt Clay pH(water) Total N P2 O5(available) K2 O(available) Organic C Total limestone
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Value 16% 58% 27% 15% 7.4 1.4 g kg−1 6.5 mg kg−1 109 mg kg−1 12 g kg−1 560 g kg−1
×100
(1)
atom% N excesssample = atom% Nsample −0.3663 atom%15 N in air (2) fixed N kg ha−1 = sainfoin N × (Ndfa /100) (3) 15
15
This method provides an accurate estimate for Ndfa because it compares 15 N enrichment in the sainfoin and in the non-fixing species; it is not directly influenced by the levels of DM or total N yield. A rate of 4 kg N ha−1 of 15 N-enriched fertilizer (10 atom% 15 N-enriched ammonium sulfate) was applied to a 3 m2 area of non-fixing reference species and sainfoin within each experimental unit. The area (3 m2 ) with the enriched 15 N was the same area sampled for the DM yield determination. The biomass samples from these areas were dried to a constant weight in a forced air oven at 65 ◦ C, air equilibrated, weighed and ground finely enough to pass through a 1 mm mesh. The dry biomass material was subjected to elemental analyzer isotope ratio mass spectrometry at the INRA UCBN Unite´ Mixte de Recherche laboratory (Caen,
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Figure 1. Percentage partitioning of phytomass as dry weight per plant at different phenological phases in sainfoin (sa) and sulla (su).
France) to determine both N content (%) and 15 N (atom%). The results were compared with those already published for fixed N in sulla,21 which were obtained from an analogous experiment carried out at the same location over the same two years using the same methodology. In fact, sulla can be considered the closest perennial forage legume to sainfoin, with identical growth cycle and production pattern. Statistical analysis For each experiment, statistical significance was determined by two-way analysis of variance (ANOVA). Differences between means were assessed by the least significant difference (LSD) test for separation of means between and within species in the experiment on polyphenolic accumulation at different phenological phases and in different plant components. In the N fixation experiment the LSD test was performed for separation of means between species. The significance level was fixed at P ≤ 0.05 for all statistical analyses.
RESULTS AND DISCUSSION Total phenolics and condensed tannins The percentage partitioning of sainfoin and sulla phytomass based on dry weight per plant showed that the contribution of each
component was similar during the examined phenological phases (Fig. 1). Statistically significant differences in average TotP concentration at different phenological phases were found in leaves and stems of sainfoin and in leaves of sulla (Table 2). Comparing the total phenolic concentrations of the two species, no significant differences were observed in leaves and stems; only racemes showed a significant difference, with sulla having a higher concentration in racemes compared with sainfoin. The average NTP concentrations in leaves, stems and racemes of sainfoin were higher than those of sulla and significantly different between phenological phases in leaves and stems of sainfoin and sulla but only in racemes of sulla (Table 3). Statistically significant differences in average CT concentration at all phenological phases were found in all components of sainfoin but only in racemes of sulla (Table 4). Comparing the CT concentrations in leaves of the two species, sulla almost always showed a higher concentration except at flowering, when no statistical difference was seen; the same trend was observed for racemes. The concentration of CT per plant is shown in Fig. 2. For each species the values were always in the range considered beneficial for animals, not exceeding 50 g DE kg−1 DM.22 The average CT concentration in sainfoin was lower than that in sulla. Variations in TotP, NTP and CT contents at different phenological phases and in different plant parts were found by several authors in sainfoin and sulla.5,10,16,23,24 Leaves were the organs with the highest TotP, NTP and CT contents for both sainfoin and sulla, and, according to the phytomass partitioning (Fig. 1), the percentage of leaves was higher at the phenological phase of flower bud in both species, while in the next phases the percentage of leaves decreased, with a marked drop in sulla at early seed set due to the high percentage of stems. The TotP, NTP and CT average concentrations in leaves of sainfoin showed an increase at the flowering phase. According to Frutos et al.9 and Gebrehiwot et al.,10 comparisons with other published CT data for similar species are very difficult owing to variations in the methods, procedures and standards used for analyses. According to Piluzza and Bullitta,11 the Folin–Ciocalteu and butanol/HCl/Fe3+ assays remain the most practical methods for screening large numbers of different plant species for TotP, NTP and CT; it is appropriate to use delphinidin as a standard for CT determination, because this substance was the most abundant constituent from the acid-catalysed cleavage of CT from the tested plant species sainfoin and sulla.11 By the use of this standard, our data are made comparable and underestimation and overestimation are avoided.11,12
Table 2. Average concentration of total phenolics (g GAE kg−1 DM) in sainfoin and sulla at different phenological phases Leaves Phase Flower bud Flowering Early seed set Spring regrowth Autumn regrowth LSD0.05 (within species) LSD0.05 (between species)
Sainfoin
Stems Sulla
40.2 54.5 44.9 48.9 56.5 6.8
40.9 48.7 48.8 46.8 NA 4.3 NS
Sainfoin
Racemes Sulla
19.4 12.9 14.1 13.5 21.8 4.4
17.6 17.5 13.5 14.7 NA NS NS
Sainfoin
Sulla
— 26.9 28.3 30.8 — NS
— 46.8 41.2 50.1 — NS 17.0
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NA, not available; NS, not significant.
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Table 3. Average concentration of non-tannic phenolics (g GAE kg−1 DM) in sainfoin and sulla at different phenological phases Leaves Phase Flower bud Flowering Early seed set Spring regrowth Autumn regrowth LSD0.05 (within species) LSD0.05 (between species)
Sainfoin
Stems Sulla
13.6 16.8 12.7 13.3 15.3 1.5
6.1 9.4 6.5 5.9 NA 0.9
Sainfoin 9.1 7.0 7.1 7.1 5.6 1.0
7.1
Racemes Sulla
Sainfoin
2.3 3.6 2.4 2.7 NA 0.2
Sulla
— 9.4 8.5 9.6 — NS
4.8
— 5.1 4.6 6.8 — 0.8 3.7
NA, not available; NS, not significant.
Table 4. Average concentration of condensed tannins (g DE kg−1 DM) in sainfoin and sulla at different phenological phases Leaves Phase Flower bud Flowering Early seed set Spring regrowth Autumn regrowth LSD0.05 (within species) LSD0.05 (between species)
Sainfoin
Stems Sulla
22.6 34.9 21.3 24.0 28.3 4.4
31.6 37.7 32.9 33.8 NA NS 8.3
Sainfoin
Racemes Sulla
10.0 5.3 8.3 5.7 10.2 2.6
Sainfoin
11.7 13.2 8.2 12.9 NA NS
Sulla
— 15.3 9.3 13.8 — 4.5
4.2
— 19.5 25.6 29.8 — 1.7 12.3
NA, not available; NS, not significant.
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Figure 2. Whole plant average concentration of condensed tannins in sainfoin and sulla at different phenological phases.
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Table 5. Aerial DM yield (kg ha−1 ) of sainfoin and non-fixing reference species Date of cutting
Sainfoin
Chicory
Oats
Table 7. Proportion of N derived from atmosphere (Ndfa ) for nonfixing reference species and fixed N in aerial biomass of sainfoin compared with fixed N in aerial biomass of sulla
LSD0.05
Fixed N (kg ha−1 )a
Ndfa (%) Spring 2002 Autumn 2002 Spring 2003 Total
2.9 1.9 3.0 7.8
0.2 3.1 7.8 11.1
7.7 — 5.6 13.1
1.3 0.8 2.6 4.2
The dynamics of CT concentration in growing plants is an important aspect for implications on ruminant nutrition and health. According to the literature, only defined concentrations of forage CT (20–50 g kg−1 DM) can be useful to increase the efficiency of protein digestion and productivity in forage-fed ruminants without depressing rumen fibre digestion or voluntary intake, and to provide cheaper and safer alternatives to the use of synthetic compounds for controlling some diseases under grazing.25 According to Guglielmelli et al.,6 feeding ruminants forage containing CT may also offer potential benefits for depressive effects on rumen methane emissions. Our CT concentrations measured utilizing a delphinidin standard at comparable stages are in the range 23–35 g DE kg−1 DM for sainfoin and in the range 32–38 g DE kg−1 DM for sulla. Taking into account the different method and standard utilized by Guglielmelli et al.6 for CT quantification, it seems anyway that the concentrations we detected might be considered potentially suitable for the reduction of methane emissions by ruminants. Nitrogen fixation potential Aerial DM yield The mean aerial DM yield of sainfoin was higher in the two spring samplings than in the autumn sampling and the contribution of sainfoin to the total biomass represented about 70, 66 and 53% at the three consecutive samplings respectively (Table 5). Sainfoin was not very competitive against weeds. The aerial yields were comparable to those obtained in some other locations in Italy26 – 28 but lower than those obtained at two Italian sites by Borreani et al.16 and Martiniello and Ciola.29 According to Canu,30 a sainfoin crop grown at the same time in a similar environment of Sardinia produced up to 8 t DM ha−1 at the spring cutting, with a contribution of sainfoin to the total DM exceeding 80%. Nitrogen content and 15 N excess of aerial DM The N concentration in sainfoin aerial DM ranged from 2.6 to 4.7% and the level was almost doubled in autumn compared with the spring harvests (Table 6). In fact, in autumn the plant material was mainly represented by leaves, leading to a corresponding crude protein value close to 30%. The 15 N excess of sainfoin aerial DM ranged from 0.0337 to 0.0533 atom% and was significantly lower
Date of cutting Chicory Spring 2002 Autumn 2002 Spring 2003
Oats
LSD
77.7 — 87.9
NS — NS
82.8 83.3 86.7
Sainfoin
Sullab
61.1 ± 4.6 35.6 ± 4.4 74.1 ± 4.8 78.4 ± 7.8 77.4 ± 17.0 151.4 ± 15.0
NS, not significant. a Values are mean ± standard error. b According to Sulas et al.21
than that detected in all samplings of both reference species owing to its dilution with atmospheric N. Nitrogen fixation The two non-fixing reference species generated very similar Ndfa values in spring 2003 and no significant difference was found between them (Table 7). Oats, being an annual reference species, did not allow autumn cutting measurements for comparison. Only chicory was considered further for the quantification of aerial fixed N. The Ndfa achieved by sainfoin, based on chicory as a reference species, ranged from 82.8 to 86.7% and was in agreement with that of other forage legumes such as alfalfa and white clover.31 In spite of the plentiful available data regarding the effects of different strain inoculations in sainfoin and its N fixation under glasshouse conditions estimated by acetylene reduction assays, very little information an Ndfa values of sainfoin obtained in the field by isotopic methods is available.13,14,32 – 34 Provorov and Tikhonovich15 reported an Ndfa value of 80% determined using the isotope dilution method under Russian conditions. In a biodiversity experiment in Germany, Roscher et al.35 reported Ndfa values ranging from about 65 to 75% obtained using the natural abundance method. Sainfoin accumulated 212.6 ± 12.2 kg ha−1 (mean ± standard error) of aerial fixed N during the two-year experiment (Table 7), and the amount of fixed N in autumn, when the N concentration reached about 5% (Table 6), was similar to the spring amounts. To our knowledge, these results represent a first estimate of the potential N fixation of sainfoin obtained in rain-fed Mediterranean conditions of southern Italy. The N fixed by the sainfoin in this experiment was below the usual range of 130–160 kg ha−1 per year reported by Provorov and Tikhonovich15 and quite far from the potential value of 270 kg ha−1 per year proposed by the same authors. Since such data arise from non-Mediterranean areas and neither the sainfoin genotype nor the rhizobial strain was reported, comparisons cannot be considered appropriate. However, the Mediterranean perennial legume sulla accumulated
Table 6. Concentration of N and 15 N excess in aerial biomass of sainfoin and non-fixing reference species 15
N (%) Date of cutting
2.6 4.7 2.9
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Chicory 2.2 2.2 1.8
Oats 1.1 — 0.9
LSD 0.01 0.09 0.07
Sainfoin
Chicory
0.0429 0.0533 0.0337
0.2828 0.3208 0.2994
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Oats 0.1971 — 0.2830
LSD 0.0181 0.0053 0.0298
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Spring 2002 Autumn 2002 Spring 2003
Sainfoin
N excess (atom%)
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Mediterranean environments also by means of trials in multiple locations.
ACKNOWLEDGEMENTS Thanks are due to the technicians Maddalena Sassu, Salvatore Nieddu, Daniele Dettori, Piero Saba and Anton Pietro Stangoni for the technical assistance provided.
REFERENCES
Figure 3. Relationship between fixed N and DM yield in sainfoin.
265 kg ha−1 of aerial fixed N (Table 7), 25% higher than that of sainfoin, at the same location in the same two-year period.21 This difference was mainly due to the difference in DM yield, which reached 12.4 t ha−1 in sulla and was 29% higher than in sainfoin during the same two years. In fact, a strong relationship exists between legume DM yield and fixed N, as the latter is calculated on the basis of DM yield. Nevertheless, to allow full exploitation of the sainfoin crop, its below-ground N, not considered in this experiment, should be taken into account. In fact, the below-ground N includes N of visible roots and N of rhizodeposits from root exudates, sloughedoff root tissue and dead roots and its contribution to the total plant fixed N may even be greater than that present in the legume aerial biomass.36,37 Relationship between fixed N and DM yield The regression of aerial fixed N on aerial DM in sainfoin showed a relationship of 22 kg fixed N t−1 aerial DM (Fig. 3), in agreement with the performances of white clover and other legume pure stands across a broad range of latitudes and about 20% higher than that achieved by a monoculture of sulla at the same location in the same two-year period.21,38 Compared with sulla, this relationship has probably been influenced by the consistent amount of nonlegumes (mainly grasses) present in the sainfoin plots, where they represented about 30–50% of the total DM. In fact, grasses compete strongly for soil N and, in their presence, legumes are forced to rely heavily on N fixation as an N source.39 On the other hand, the same consistent amount of non-legumes negatively affected the performance of sainfoin in terms of fixed N per hectare.
CONCLUSIONS
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Sainfoin field grown under rain-fed Mediterranean conditions showed beneficial CT concentrations that can be considered suitable to contribute to the improvement of animal performance and health. From our results it appears that sainfoin could be comparable to and as efficient as sulla, having equal or higher N fixation potential. The favourable CT contents and the N fixation potential suggest that sainfoin can enlarge the very limited set of perennial leguminous species suitable for the organization of Mediterranean rain-fed forage systems. For these reasons the genetic potential of sainfoin should be better exploited in
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