Effect of Rhizobium Inoculation on Herbage Yield, Quality and Nitrogen Fixation of Annual Forage Legumes on Nitisols in Central Highlands of Ethiopia

ABSTRACT

The study was conducted with the objectives to evaluate the effect of Rhizobium leguminosarum biovar. viciae strain V6005 inoculation on yield, feed quality and nitrogen fixation of hairy vetch (Vicia villosa Roth.), woolly-pod vetch (Vicia dasycarpa Roth.), common vetch (Vicia sativa L.) and narbon vetch (Vicia narbonensis L.) on Nitisols at Holeta in the central highlands of Ethiopia. The experimental design was a randomized complete block design with three replicates. Analysis of variance (ANOVA) indicated that Rhizobium inoculation significantly (p < 0.05) affected vigor, root length, Root Dry Matter (RDM) accumulation, number of nodules per plant, crude protein (CP) and nitrogen fixation of these legumes. However, inoculation did not significantly (p > 0.05) influence days to emergence, stand count, plant height, fresh biomass yield, nodule fresh weight, nodulation rate, Neutral Detergent Fiber (NDF), Acid Detergent Fiber (ADF), ash, Organic Matter Digestibility (OMD) and Dry Matter (DM). Under low temperature characteristics of the study site woolly-pod vetch and hairy vetch showed a more promising agronomic performance and nitrogen fixation than common vetch and narbon vetch. The order of nitrogen fixation potential was woolly-pod vetch > hairy vetch > common vetch > narbon vetch as inoculated treatments of the respective species fixed 197.94, 165.80, 41.63 and 35.56 when compared to uninoculated control where these rates were 126.57, 127.64, 23.46 and 27.25 kg N ha^-1. Improvement in nitrogen fixation of 22.40%, 34.85%, 74.78% and 39.69% was recorded due to inoculation for hairy vetch, woolly-pod, common vetchand narbon vetchrespectively. Crude protein content of inoculated species was also improved following the same trend as for nitrogen fixation. This study indicate that the higher nitrogen yield obtained from tested Vicia species due to Rhizobium inoculation, especially from woolly-pod and hairy vetches depicts potential contribution of effective rhizobial strains to improve soil fertility and feed quality under subsistent mixed crop-livestock farming systems of the Ethiopian highlands.

 

Keywords:  Forage legumes, Rhizobia, Inoculation, Nitisols

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Introduction

The importance of forage legumes as a feed lies primarily in their high protein content (5.6-35.8 %) (Abbeddou et al., 2011; Larbi et al., 2011; Lithaourgidis et al., 2006; Mpairwe et al., 2003). Moreover, their ability to fix atmospheric nitrogen (N₂) better than food legumes also contributes positively towards the nitrogen (N) balance of the soil (Zahran, 2001; Reo and Mathuva, 2000). A review made by Nnadi and Haque (1986) reported root N-content of forage legumes ranges from 15 – 115 kg N ha^-1. But the amount of N left in the roots appears not to be rather genetically determined than related to the total N fixed. Woolly-pod vetch, hairy vetch, common vetch and narbon vetch, which are not indigenous to Ethiopia, are valuable sources of protein and minerals for small-scale livestock production systems that are often subject to inadequate or poor quality feed (Assefa et al., 2011; Assefa et al., 2006). These crops can be easily integrated into the farming systems of low input crop-livestock mixed agriculture in the highlands of Ethiopia. Moreover, use of these crops on fallow lands is a low cost method of improving soil fertility and organic matter. Legumes also have the ability to rehabilitate degraded land by improving the physical, chemical and biological characteristics of soil (Zahran, 2001). Hence, their contribution to small-scale farming systems is a key factor in sustaining long-term fertility in mixed crop-livestock production systems, especially in developing countries like Ethiopia where agricultural activities largely depend on soil nutrient reserves.

Many agricultural soils in Ethiopia are rather infertile and inherently support only low productivity agriculture. To bring about economically viable agricultural production, a high input of fertilizer (especially N and P) is essential. However, despite the potential for increasing yields and farm income by the use of fertilizers, many small-scale and poor farmers do not have the resources to use them. Furthermore, in modern agriculture, the replenishment of soil N by extensive application of chemical fertilizers has several negative environmental impacts (De Jong et al., 2008; Liu et al., 2005a). However, BNF via legume-Rhizobium symbiosis is a potential option which is an environmentally friendly, economically viable and renewable source of N for resource poor farmers (Bekere & Hailemariam, 2012; Hailemariam & Tsige, 2006, Zahran, 2001). With BNF, a large proportion of N accumulated in forage legumes is available for fertilizing the soil which is not possible with food legumes by which most N is removed at their harvest (Reo & Mathuva, 2000).

For N₂ fixation, legumes enter into symbiosis with their specific nodule forming bacterial partner, i.e. rhizobia. Too often soils neither have the proper kind of nodule forming bacteria nor sufficient indigenous rhizobial populations to meet the requirements for establishing symbiosis with their legume crops (Adjei & Chambeiss, 2002). Although inoculation of legumes is generally recommended to maximize their potential for nodulation and N₂ fixation (Bulter & Evers, 2004; Jennings, 2004; Giller, 2001), very limited work has been done on agriculturally important forage legumes in Ethiopia. According to Getahun et al., (2002), about 119 species of herbaceous forage legumes were tested in Ethiopia at about 40 testing sites of variable different agro-ecologies. Out of these species about 82% (98 species) were either exotic or commercial species introduced from various parts of the world. In Ethiopia, different species and varieties of indigenous and introduced forage legumes were evaluated and the most promising materials are used in different agro-ecologies of the country (EARO, 2000; Gebrehiwot & Tadesse, 1985). However, the complementary selection and introduction of the corresponding legume specific rhizobia has not been done yet.

Success of rhizobia inoculation is highly site specific and depends on a multitude of interactions including environmental, soil and biological factors (Argaw, 2012;

Lira Junior et al., 2005). Accordingly, there is a critical need to evaluate the contribution of inoculation on forage legumes in the farming system of the central Ethiopian highlands. Therefore, the present study was conducted to determine the effect of Rhizobium inoculation on nodulation, N₂ fixation, yield and forage quality of selected annual forage legumes (Vicia species selected based on their agronomic performance) growing on Nitisols in the central highlands of Ethiopia.

 

Materials and Methods

Study Area

The study was carried out at Holeta Agricultural Research Center (in Welmera), Southwest Shewa zone of Oromia Regional State, Ethiopia (Figure 1) which is situated at an altitude of 2400 m.a.s.l.; 9^O 3′ N 38^O 30′ E with 1055.02 mm mean annual rainfall; 6.1 ^OC mean minimum temperature and 22.2 ^OC mean maximum temperature. The experimental site is located at about 30 km west of Addis Ababa along the road to Nekemte. The soil type in the area is predominantly Nitisols which is characterized by well-drained soil with diffuse horizon boundaries and surface horizon containing more than 30% clay (Mekonnen, 2007). Our result also shows that the sand (17.5%), silt (15%) and clay (67.5) contents put the soil under clay textural class (USDA). Following classifications of Landon (1991), the soil of the study site can further be characterized as follows: acidic in pH; very low in Organic Carbon (OC) and N; medium in available Phosphorus (P) and Cation Exchange capacity (CEC) and high exchangeable bases. Bulk density (1.29) and porosity (0.53) of the soil are good for agriculture. The particle density (2.74) also shows that the soil is non-ferruginous and non-humus. Soil acidity and low nutrient content may limit the productivity and the range of species in the study area (Table 1). The farming system in the study area is mixed crop-livestock production system where barley (Hordeum vulgare L.), bread wheat (Triticum aestivum L.), teff (Eragrostis tef (Zucc.) and potato (Solanum tuberosum L.) are the major growing crops while cattle, sheep, goat, equines (horse and donkey), poultry (chickens) are major livestock species supporting the livelihood of the farming community.

Experimental Design and Treatments

The experiment was conducted during the cropping season of the main rainy season in 2010 (June-September). The treatments were applied to four species of forage legumes and two inoculation systems (with and without inoculation). Forage legumes were hairy vetch (Vicia villosa Roth.), woolly-pod vetch (Vicia dasycarpa Roth.), common vetch (Vicia sativa L.)and narbon vetch (Vicia narbonensis L.).  Additionally, oat (Avena sativa var. lampton) was included as non-nitrogen fixing reference crop to determine the amount of N₂ fixed by these forage legumes using the difference method. Factorial combinations of the nine treatments (uninoculated Vicia species, inoculated Vicia species and oat (non-nitrogen fixing reference crop)) were laid out in a randomized complete block design with three replicates. The experimental plots measured 2.4 m by 3 m (7.2 m²) and a spacing of 1.5 m was maintained between plots. Blocks were spaced 2 m apart and a buffer zone of at least 2 m was maintained around the experimental area.

Culture of Rhizobium leguminosrum biovar viciae, strain V6005 was used for inoculation of different Vicia species (forage legumes). Seeds of inoculated forage legumes were first coated with gum arabic by mixing 1000 g of seed with 30 ml of gum arabic solution (40 g gum arabic per 100 ml distilled water). When all the seeds were wet, 80 g of the carrier material (filter mud from sugar factory) were added and the seeds were remixed until the seeds were well coated. The seeds were then allowed to dry at room temperature (~ 20 ^OC) for 30 minutes. Before planting the inoculated seeds were sheltered from heat and radiation. The row spacing for all Vicia species used in this study was 30 cm while the reference crop (oat) was planted at row spacing of 20 cm. Seeds of hairy vetch, woolly-pod vetch, common vetchand narbon vetch were planted by hand in rows. Seed rate of hairy vetch, woolly-pod vetch and common vetchwere 30 kg ha^-1 while narbon vetch were planted at seed rate of 100 kg ha^-1. The reference crop (oat) was planted at seed rate of 80 kg ha^-1. A starter dose of 18/46 N/P₂O₅ kg ha^-1 was applied for all treatments at plating. During planting, uninoculated seeds of the Vicia species were sown first on their plots and covered with soil. Inoculated seed of Vicia species were then sown on their respective plots and covered with the soil. On space between block (2 m) and plots (1.5 m) furrows with 25 cm width and 30 cm depth were prepared on either side of all plots to avoid plot to plot contamination. These furrows were regularly monitored and refreshed by removing soils refilled by rainfall induced water erosion.

The rhizobial inoculant (Rhizobium leguminosarum biovar vciae, strain V6005) used in this study was isolated from soils in the central highlands of Ethiopia (Welemra and Dendi areas, figure 1)  where these and other related legumes such as grass pea (Lathyrus sativus L.) were widely cultivated. V6005 (indigenous rhizobia) was selected from soils tested (rhizobial population and nodulation of Vicia species) following method described by Somasegaran & Hoben (1985). This strain (V6005) which was confirmed as a rhizobia by the ability to form nodules on Vicia species was used to evaluate their effect on agronomic, quality and nitrogen fixation of selected forage legumes (Vicia species).

Data Collection

Soil Sampling and Analysis

Soil samples from each plot were collected randomly at a depth of 0-30 cm (plow layer) by using an auger. Samples were collected for two time points: just before sowing and at harvesting (50% flowering). Three composite samples (one under each block) were prepared from soils sampled randomly from each plots before sowing. During composite sample preparation, soil samples collected randomly from each plot in a block were mixed thoroughly in a bucket and subsample (500 g) were taken. Soil samples were then air dried to a constant weight, ground and sieved to pass a 2 mm mesh before analysis. The soil samples were analyzed for selected physical and chemical properties as described below. Results of analysis (soil chemical properties) from samples collected before sowing were used as control treatments together with results of soil analysis obtained from samples collected randomly from each plot at harvest to see soil nutrient dynamics under each treatment. Soil physical properties were determined from samples taken before sowing.

The soil texture was measured by using the Bouyoucos hydrometer method (Bouyoucos, 1962). Bulk density was determined from undisturbed soil that was taken with a core sampler of known dimension from a soil depth of 0 – 30 cm as described by Robert et al. (2002). The pycnometer method was followed in determining soil particle density as described by Robert et al. (2002). The estimation of total porosity was calculated based on the values of bulk density (r_b) and particle density (r_p) following the equation (Millar et al., 1965):

Total porosity = 1-  (ρ_b/ρ_{p )}    

Where:  r_b= bulk density; r_p= particle density

 

Soil Chemical Properties

Soil pH was determined using the pipette method (Day, 1965). Soil organic carbon (OC) was determined as described (Allison, 1960), soil and plant nitrogen (TN) was obtained using the Kjeldahl method (Ransit et al., 1999). Cation Exchange Capacity (CEC) was determined by ammonium acetate saturated samples through distillation and measuring the ammonium content using a modified Kjeldahl procedure (Ranist et al. 1999); available phosphorus (Pav) was measured using NaHCO₃ extraction method (Olsen et al., 1965) and an UV/vis spectrophotometer. Exchangeable bases were determined after saturating the soil with 1M ammonium acetate. Ca²⁺, Mg²⁺ values were determined using an atomic absorption spectrophotometer while K⁺ was determined using a flame photometer as described by Robert et al. (2002).

Plant Analysis

Bulk plant samples were taken at a 50% flowering stage of each species and oven dried at 65 ^OC. The samples were ground and analyzed for total nitrogen (TN) using Kjeldahl’s method. Dry Matter (DM), Crude Protein (CP), fiber and ash were also determined as described in  AOAC (1990) method and the amount of nitrogen (N₂) derived from the atmosphere was determined by comparing the nitrogen accumulation of the legumes and the non-legume reference crop (oat, cv. lampton) (Hardarson & Danso, 1993).

Forage Legumes’ Agronomic Performance, Nodulation and N₂ fixation

Days to 50% emergence, vigor, plant height (unstretched), fresh and dry matter yields of each treatment were recorded. At 50% flowering five samples were taken randomly from each plot using a 50 cm x 50 cm quadrant for determination of dry biomass yield. The fresh weight of the sampled plants was determined then the plants were oven dried at 65 ^OC for 72 hours to determine dry matter as described in AOAC (1990). At the same stage (50% flowering) when samples were taken for dry biomass yield determination, 10 plants were selected randomly from each plot and uprooted with their soils for nodulation measurement. Soils on roots of each plant were carefully removed manually by dissolving the soil with clean water. Roots were washed for nodulation study following Pal & Saxena’s (1975) method. Nodule number, dry weight and nodulation rating were recorded. Nodulation rating was done by examining nodules in the taproot (primary root), in the secondary roots  close to the taproot, by examining the entire root system and by examining plants showing no root nodulation. The rating of the plants for nodulation was done according to a scale of 1-10. The number of plants, which have developed nodules on the taproot, close to the taproot, or scattered over the entire roots and plants with no nodules on their roots were identified and subjected to the following formula for nodulation rating (NifTAL, 1979).

Nodulation rating=  ((10*NPTRN)+ (5*NPNCTR)+ (1*PSN)+ (0*PNN))/n

Where,

NPTRN: Number of plants with taproot nodulation

NPNCTR: Number of plants with nodules close to taproot

PSN: Number of plants with scattered nodulation

PNN: Number of plants without nodulation

n: Total number of plants

Nitrogen fixation of these forage legumes was estimated by comparing the N accumulation of the legumes and non-legume reference crops (Oat var. lampton) (Hardarson & Danso, 1993; Peoples & Herridge, 1990). The reference crop was selected with the assumption that the legumes and the oat variety exhibit similar characteristics as far as the rooting volume and the ability to extract N and to accumulate soil N are concerned. Selection of reference crop is crucial as the method for measuring the N₂ fixation by legumes relay on N uptake by a non-N₂-fixing plant as a surrogate for uptake of soil N by a N₂-fixing legume. To determine the quantity of N derived from N₂ fixation, the procedure as described by Peoples et al. (1989) was applied in a modified way. The differences in post-harvest soil mineral N in the fixing and non-fixing plots are added to the differences in total N yields of the two crops as follows:

Q= [N yield (legume)- N yield (Control)]+ [N soil (legume)+ N soil (control)]

Where,

Q: N₂ fixed by legume

N: Crop nitrogen

 

Statistical Analysis

Data were analyzed using the Statistical Analysis Software (SAS Institute, Inc., Carry, NC, USA) to perform ANOVA (SAS 9.1, Proc. GLM) in a randomized complete block design. Means of all treatments were calculated and the difference was tested for significance using the least significant difference (LSD) test at p=0.05.

Statistical model is:

Yijk = m + t_i + b_j  + tb_(ij) + e_ijk

Where:

m – grand mean

t_i – the effect of i^th Vicia species (i = 1 to 4)

b_j– the effect of j^th inoculation (j = 1 to 2)

tb_(ij)– interaction effect of the combination ij

e_ijk – random error (0, d²)

 

Results

Soil chemical properties response to inoculation

In attempt to assess changes in chemical properties of the soil pH, percent nitrogen (% N), available phosphorus (Pav) and Cation Exchange Capacity (CEC) of the soil before sowing and at harvest of Vicia species (forage legumes) were obtained to vary significantly (P < 0.05). However, organic carbon (OC),  sodium (Na), calcium (Ca) and magnesium (Mg) content of the soil before sowing and at harvest did not vary significantly (P > 0.05) (Table 1). Only the pH of soil at harvest on which inoculated woolly-pod vetch was planted increased significantly (P £ 0.05) compared to soil pH before sowing. On the other hand, nitrogen content (% N) of the soil on which inoculated hairy vetch was planted showed a significant increase (P = 0.02) over soil N content before sowing. Unlike other soil nutrients, more variability was observed in soil available phosphorus (Pav) before sowing and at harvest under different forage legumes and inoculation treatments.  P content of the soils at harvest under oat (non-legume), uninoculated narbon vetch and uninocuated hairy vetch were higher (P £  0.05) than soil P before sowing. However, the P content of the soil under both inoculated and uninoculated woolly-pod and hairy vetch did not vary (P > 0.05). Cation Exchange Capacity of the soil at harvest varies (P £ 0.05) only under uninoculated narbon vetch (reduced) compared to the soil CEC before sowing. Generally the soil in the study area was acidic in pH; low in OC and N; medium in available P and CEC; and high in exchangeable bases (Landon, 1991).

Days to Emergence, Vigor and Stand Count

Days to emergence, vigor and stand count significantly vary (p < 0.05) among the species (Table 2). Regarding the days to emergence, narbon vetch and common vetch were established faster than hairy and woolly-pod vetches. Among the tested species narbon vetch had the largest seed size which might contributed to early establishment. Vigor of hairy vetch was better than others and significantly higher (p < 0.05) than narbon vetch. However the stand count after establishment of hairy vetch was significantly (p = 0.003) lower than narbon vetch and common vetch. Rhizobium inoculation affected the growth (vigor) of Vicia species significantly (p = 0.004), while days to emergence and stand count after establishment were not affected (p > 0.05) by inoculation (Table 2). Inoculation tends to improve the vigor of all species tested. However, specific to a species only common vetch responded significantly to inoculation. Among the treatments, vigor of inoculated hairy vetch was significantly higher than both inoculated and uninoculated common vetch and narbon vetch. Generally, days to emergence and stand count were influenced by the species while vigor was influenced both by species and inoculation where hairy vetch responded better than other species.

Plant Height and Biomass Yields

Plant height was significantly (p < 0.0001) different between the legume species in which woolly-pod vetch was the tallest followed by hairy vetch, narbon vetch and common vetch (Table 3). This variation in plant height of these forage legumes has an agronomic importance for integration and production with different food crops. Inoculation had no significant (p = 0.39) effect on plant height (Table 3). Fresh and dry biomass yields of evaluated forage legumes did vary (p-values of 0.31 & 0.19 respectively) owing to Rhizobium inoculation (Table 3). However, above ground fresh (p = 0.001) and dry matter (p = < 0.0001) yields of the forage species vary significantly. Woolly-pod vetch yielded the highest amount of fresh and dry biomass while common vetch yielded the lowest. Inoculated woolly-pod vetch gave the highest fresh and dry biomass yields.

Root Length and Dry Matter Accumulation

Root length (p = 0.005) and root dry matter (p = 0.03) accumulation of evaluated forage legumes were very significantly influenced by Rhizobium inoculation. Common vetch responded significantly to inoculation and its root length was superior over all other inoculated and uninoculated treatments (Table 4). Although root lengths of inoculated treatments of all species were higher than the uninoculated ones, response to inoculation by hairy vetch, woolly-pod vetch and narbon vetch were not significant. Similarly the species difference had also a significant (p = 0.0004) effect on root length. The highest root length was observed from common vetch while the least was recoded from narbon vetch. Similar to the effect on root length, Rhizobium inoculation increased RDM accumulation of forage legumes. Hairy vetch responded to inoculation more readily than other species. RDM accumulation was also significantly (p = 0.002) different between species, especially hairy vetch and woolly-pod vetch gained more DM than the other tested legumes.

 

Fig. 1. Map of the study area (Welmera) and Rhizobium collection sites (Dendi and Welmera), Southwest Shewa zone , Oromia, Ethiopia

 

Note: Nfix I- = N₂ fixed by uninoculated species; Nfix I+ = N₂ fixed by inculated species

Fig. 2. Nitrogen fixed by forage legumes (Vicia species) in response to  Rhizobium inoculation

 

Nodulation

Number of nodules per plant, nodules fresh weight, nodule dry matter accumulation and nodulation rate are presented in table 5. Among the nodulation parameters Rhizobium inoculation significantly influenced (p = 0.02) only the number of nodules per plant while nodule fresh weight, nodule dry weight and nodulation rates were not affected (p > 0.05). Inoculated common vetch showed the highest number of nodules per plant in both inoculated and uninoculated treatments. Although the number of nodules per plant from common vetch was high, visual inspection showed that the size of the nodules was small. Generally, it seems that it is not only the number of nodules but also their size which influence size which influence the dry matter accumulation. However all parameters: number of nodules per plant (p = 0.0003), nodules fresh weight (p = 0.04), nodules dry matter accumulation (p = 0.002) and nodulation rate (p £ 0.05) vary significantly among the legume species.

 

Table 1. Soil chemical properties at planting and harvest of Vicia species with and without Rhizobium inoculation

Soil samples from plots under	pH	%N	P	OC	Na	K	Ca	Mg	CEC
Vicia villosa I+	5.25b	0.227a	4.60abc	1.73	1.38	5.58	29.17	13.43	18.41ab
Vicia villosa I–	5.38ab	0.207bcd	3.07bc	1.62	1.29	4.89	29.87	13.61	18.29ab
Vicia dasycarpa I+	5.27b	0.213abc	4.33abc	1.65	1.93	5.04	32.44	13.81	17.45ab
Vicia dasycarpa I–	6.68a	0.200bcd	5.20ab	1.63	1.37	5.00	30.37	13.33	17.86ab
Vicia sativa I+	5.44ab	0.217ab	4.90abc	1.73	1.85	5.04	30.93	13.61	19.18a
Vicia sativa I–	5.26b	0.193d	3.53abc	1.55	1.39	4.73	30.57	13.56	17.36ab
Vicia narbonensis I+	5.29b	0.197cd	3.60abc	1.51	1.34	4.73	30.69	13.65	15.88b
Vicia narbonensis I–	5.38ab	0.207bcd	3.30bc	1.58	1.72	5.28	29.71	13.43	18.92a
Oat (non-legume)	5.44ab	0.190d	3.00c	1.62	1.39	4.32	31.03	13.45	17.51ab
Control*	4.94b	0.200bcd	5.60a	1.79	1.61	5.03	29.50	13.75	18.24ab
Overall Mean	5.43	0.20	4.11	1.64	1.52	4.96	30.43	13.56	17.91
Mean at harvest**	5.48	0.21	3.95	1.62	1.52	4.95	30.53	13.54	17.87
CV (%)	14.26	5.37	31.09	10.54	25.58	18.47	10.73	4.11	8.89
LSD	1.33	0.019	2.19	NS	NS	NS	NS	NS	2.73

*Control = Composite soil samples taken from each block at planting and used as a comparison for soil chemical properties at harvest. **Mean at harvest =Mean values of the parameters excluding the control (samples at planting)

Note: I⁺ =Inoculated; I^– =Uninoculated; CV =Coefficient of Variation; LSD =Least Significant Level;

NS =Non significant

 

Table 2. Effect of Rhizobium inoculation on days to emergence, vigor and stand count of four forage species grown on Nitisols at Holeta

Forage species	Days to Emergence				Vigor (1-5)*				Stand count ‘000 ha-1
	I–	I+	Mean		I–	I+	Mean		I–	I+	Mean
Vicia villosa	12	12	12		3.83	4.5	4.17		318.5	333.3	325.9
Vicia dasycarpa	13	12	12		3.8	4.17	3.99		333.3	357.4	345.4
Vicia sativa	10	11	10		3.33	4.17	3.75		544.4	675.9	610.2
Vicia narbonensis	9	10	9		3.33	3.83	3.58		487	538.9	512.9
Mean	11	11			3.57	4.17			420.8	476.4
Overall Mean			11				3.87				448.6
CV(%)		9.35				9.95				27.12
LSD (5%)
Species		1.28				0.51				150686
Inoculation		0.9				0.36				106551
Species x Inoculation		1.81				0.73				213102

Note: I^– Uninoculated; I⁺ Inoculated; CV Coefficient of Variation; LSD Least Significant Difference; ha hectare; *Vigor: 1- Very small, 2- Small 3- Medium, 4- Large, 5- Very large

 

Nodule fresh weight gain, dry matter accumulation and nodulation rates of common vetch were the lowest of all the tested legumes. Despite having the lowest nodule number per plant, narbon vetch gained the highest nodule dry matter. The nodulation rates of woolly-pod vetch, narbon vetch and hairy vetch were promising.

Forage Quality

Crude protein (CP) content of the legume species was highly improved (p = 0.001) due to inoculation with the Rhizobium strain while Acid Detergent Fiber (ADF), Neutral Detergent Fiber (NDF), Ash, Organic Matter Digestibility (OMD), DM did not show significant  difference (p > 0.05) (Table 6). Hairy vetch, woolly-pod vetch and common vetch significantly responded in CP composition to inoculation. CP content of inoculated hairy vetch, woolly-pod vetch, common vetch and narbon vetch was improved by 15.25%, 15.16%, 10.39% and 8.9% compared to their respective uninoculated treatments. Except ash content (p = 0.11) and organic matter digestibility (p = 0.12), NDF, ADF, hemicellulose (HC), CP, and DMD showed a significant (p £ 0.05) difference between the evaluated species.

Nitrogen Fixation

The interaction effects of Vicia species and Rhizobium inoculation on nitrogen fixation was significant (p £ 0.05). The obtained results indicate that inoculation of woolly-pod and hairy vetch significantly (p=0.0004) increased the N₂ fixation when compared to the uninoculated treatments of these species or when compared to the inoculated and uninoculated treatments of common vetch and narbon vetch. Woolly-pod vetch and hairy vetch showed the highest performance and N₂ fixation rates (Table 7). The order of nitrogen fixation potential was determined to be as follows: woolly-pod vetch > hairy vetch > common vetch > narbon vetch. Inoculated treatments of the respective species fixed 197.94, 165.80, 41.63 and 35.56 against 126.57, 127.64, 23.46 and 27.25 kg N ha^-1 when uninoculated (figure 2). This corresponds to an improvement in nitrogen fixation of 22.40%, 34.85%, 74.78% and 39.69% respectively for inoculated hairy vetch, woolly-pod vetch, common vetch and narbon vetch.

 

Discussion

Soil chemical properties

Soil environment is under a constant state of change owing to management systems.  Fluctuations in pH, N, P and CEC observed due to inclusion of forage legumes with different management in the farming system explains this fact. Legumes are well recognized as key components of low input agriculture in which land degradation due to fertility depletion is the major cause of low productivity (Atemkeng et al., 2011). Improvements in soil pH, nitrogen content (Table 1) also demonstrate the vital roles of forage legumes in soil nutrient recovery beside an important source of feed for livestock. The increased soil pH at harvest compared to the content before sowing, might be due to nitrate arrest by the roots of forage crops which decrease the rate of acidification. The increasing trend of soil nitrogen under both inoculated and uninoculated hairy vetch and woolly-pod vetch; inoculated common vetch and uninoculated narbon vetch is attributed to their ability to fix nitrogen. Particularly, significant increase of soil nitrogen under inoculated hairy vetch depicts the importance of inoculation and variation in the potential contribution of different forage legumes to the soil N economy.  Coupled to role of inoculation and specificity of the legume species to a particular strain of rhizobia, genetic potential and agronomic performances in response to local climatic variables are responsible for differences in N status of the soil at harvest.   Unlike pH and nitrogen, soil phosphorus and CEC tends to lower. Inconsistent changes in CEC in soils under tested forage legumes (with and without inoculation) might be associated with varying OC.

 

Table 3. Effect of Rhizobium inoculation on plant height, fresh and dry biomass yields of four forage legumes grown on Nitisols at Holeta

Forage species	Plant height* (cm)				Fresh biomass yield				Dry biomass yield            (t ha-1)
					(t ha-1)
	I–	I+	Mean		I–	I+	Mean		I–	I+	Mean
Vicia villosa	129.58	133.33	131.46		26.55	29.17	27.86		4.87	5.4	5.14
Vicia dasycarpa	154.83	163.89	159.36		34.45	49.26	41.86		5.45	6.68	6.07
Vicia sativa	28.22	31.22	29.72		3.57	8.2	5.89		0.94	1.46	1.2
Vicia narbonensis	44.4	52	48.2		5.48	7.92	6.7		1.46	1.74	1.6
Mean	89.26	95.11			17.51	23.64			3.18	3.82
Overall Mean			92.18				20.58				3.5
CV(%)		17.9				68.84				32.59
LSD (5%)
Species		20.44				17.54				1.41
Inoculation		14.45				12.4				0.99
Species x Inoculation		28.91				24.81				0.69

Note: I^– Uninoculated; I⁺ Inoculated; CV Coefficient of Variation; LSD Least Significant Difference; cm centimeter; t tone; ha hectare; *unstretched height

 

Table 4. Effect of Rhizobium inoculation on root length and root dry matter accumulation of four forage legumes grown on Nitisols at Holeta

Forage species					Root dry matter (g pl-1)
	Root length (cm)
	I–	I+	Mean		I–	I+	Mean
Vicia villosa	21.67	23.56	22.62		1.19	1.84	1.52
Vicia dasycarpa	20.4	20.67	20.54		1.22	1.31	1.27
Vicia sativa	21.8	26.53	24.17		0.81	0.99	0.9
Vicia narbonensis	17.67	19.8	18.74		0.75	0.87	0.81
Mean	20.39	22.64			0.99	1.25
Overall Mean			21.51				1.12
CV(%)		7.81				24.84
LSD (5%)
Species		2.08				0.34
Inoculation		1.47				0.24
Species x Inoculation		2.94				0.49

Note: I^– Uninoculated; I⁺ Inoculated; CV Coefficient of Variation; LSD Least Significant Difference; cm centimeter; pl plant

 

Table 5. Effect of Rhizobium inoculation on Nodulation parameters of four forage legumes grown on Nitisols at Holeta

Forage species	Nodules pl-1				Nodule fresh weight (mg pl-1)				Nodule dry weight (mg pl-1)				Nodulation rate
	I–	I+	Mean		I–	I+	Mean		I–	I+	Mean		I–	I+	Mean
Vicia villosa	42	56	49		328.33	425	376.67		41.6	59.67	50.64		1.8	2.97	2.39
Vicia dasycarpa	53	46	49		360.33	456.67	408.5		111.33	117.7	114.52		3.6	4.57	4.09
Vicia sativa	52	138	95		165	283.33	224.17		86	120.67	103.34		1.5	1.97	1.74
Vicia narbonensis	10	16	13		413.33	695	554.17		142	186.33	164.17		3.27	4.3	3.79
Mean	39	64			316.75	465			95.23	121.09			2.54	3.45
Overall Mean			52				390.87				108.16				3
CV(%)		45.76				46.26				36.68				53.45
LSD (5%)
Species		29.26				223.93				49.13				1.98
Inoculation		20.69				158.35				34.74				1.4
Species x Inoculation		41.38				316				69.48				2.8

Note: I^–  Uninoculated; I⁺ Inoculated; CV Coefficient of Variation; LSD Least Significant Difference; t tone; ha hectare; pl plant

 

Table 6. Effect of Rhizobium inoculation on feed compositions of four forage legumes grown on Nitisols at Holeta 

Forage species	NDF (%)				ADF (%)				Ash (%)				CP (%)				OMD (%)				DM (%)
	I–	I+	Mean		I–	I+	Mean		I–	I+	Mean		I–	I+	Mean		I–	I+	Mean		I–	I+	Mean
Vicia villosa	47.61	49.5	48.56		40	40.7	40.33		10.68	11.2	10.96		22.78	26.88	24.83		89.3	88.77	89.05		93.6	94.2	93.87
Vicia dasycarpa	49.4	50.17	49.79		41.8	41.8	41.8		12.41	12.3	12.36		21.15	24.93	23.04		87.6	87.69	87.64		92.9	93.2	93.01
Vicia sativa	44.72	40.25	42.49		36.5	34.1	35.29		12.16	12.4	12.26		21.12	23.57	22.35		87.8	87.64	87.74		93.5	93.4	93.47
Vicia narbonensis	48.81	46.78	47.8		38	38.6	38.31		10.84	11.7	11.25		17.84	19.58	18.71		89.2	88.33	88.75		93.2	92.4	92.8
Mean	47.64	46.68			39.1	38.8			11.52	11.9			20.72	23.74			88.5	88.11			93.3	93.3
Overall Mean			47.16				38.93				11.71				22.23				88.29				93.29
CV(%)		5.67				4.97				9.7				4.69				1.28				0.43
LSD (5%)
Species		3.31				2.39				1.4				1.29				1.4				0.49
Inoculation		2.34				1.69				0.99				0.91				0.99				0.35
Species x Inoculation		4.68				3.39				1.99				1.83				1.99				0.7

Table 7. Effect of Rhizobium inoculation Nitrogen fixation of four forage legumes grown on Nitisols at Holeta

Forage species	Fixed N2 (kg ha-1)
	I–	I+	Mean
Vicia villosa	170.26	290.54	230.4
Vicia dasycarpa	182.17	301.59	241.88
Vicia sativa	33.1	70.28	51.69
Vicia narbonensis	42.17	51.2	46.69
Mean	106.93	178.4
Overall Mean			142.66
CV(%)		26.69
LSD (5%)
Species		47.16
Inoculation		33.35
Species x Inoculation		66.7

Note: I^– Uninoculated; I⁺ Inoculated; CV Coefficient of Variation; LSD Least Significant Difference

 

The CEC of the soil is mainly affected by the clay content and the amount and decomposition of organic matter (Foth, 1990). The lower OC of the clay soil in the study area may lead to the point that pH has much less importance to changes in CEC as much of the CEC originate from clays. The pivotal role of P in legume crops growth and productivity is well documented (Atemkeng et al., 2011). The reduction of P in the soil after legumes growth is associated with the uptake by these plants and suggests need of P enrichments for their better growth, productivity and nitrogen fixation.

Days to Emergence, Vigor and Stand Count

Emergence is probably the single most important event that affects the success of an annual crop. Rapid uniform and complete emergence of vigorous seedlings shorten the time to complete ground cover, establishment of optimum canopy structure to minimize interplant competition and provide plants with time and special advantages to compete with weeds (Havilah, 2011). Better growth (vigor) of inoculated species (Table 2) is also suggesting that the inoculum strain was competitive with native strains for nodule sites and effective in N₂ fixation with the tested hosts (Date, 2000).

It is known that, seed size, sowing depth, land preparation and environment influences the germination, emergence and stand establishment of crops. Uniform seedbed preparation is vital to successful forage stand establishment, particularly small-seeded plants (Pal, 2004). Variations observed in days to emergence and stand count (Table 2) may be related to inherent traits which are host specific. Moreover, differences in seed germination rates of hairy vetch (67%), narbon vetch (99%), common vetch (98%) and woolly-pod vetch (95%) may account for differences in stand count of the species.

Plant Height and Biomass Yields

Many factors are known to affect plant height and herbage yield. These include species or accessions (Lopez et al., 2005; Badrzadeh et al., 2008; Firincioğlue et al., 2010), the amount and distribution of rainfall during the growing season (Abd El-Moneim and Cocks, 1993) and phosphate availability (Turk, 2007).  Our results (Table 3) are in agreement with those reports made on the effect of species on growth and yield. According to Labri et al. (2011) days to flowering; seedling vigor, tolerance to low temperature, frost, diseases and low pH are important traits for plant growth and biomass accumulation in the highland environment. This implies that woolly-pod vetch and hairy vetch have better potential for growth and biomass accumulation in cool highlands than common vetch and narbon vetch. Late flowering characteristics of woolly-pod vetch and hairy vetch compared to common vetch and narbon vetch might contribute to a higher biomass yield. A similar correlation was also reported for common vetch, narbon vetch and woolly-pod vetch (Abd El-Moneim, 1993).

Root Length and Dry Matter Accumulation

Roots are important plant organs that absorb water and nutrients from soil. They also give mechanical support to plants and supply hormones that affect many physiological and biochemical processes associated with growth and development (Fageria and Moreira, 2011). The responses of root length and dry matter accumulation (Table 4) to inoculation in these forage legumes have an implication on development of plants, yield, nitrogen cycle and microbial activity. According to Sainju et al. (2005a), roots left in the soil after crop harvest are the main contributor to N₂ cycle. The difference seen among the forage legumes is due to inherent genetic variations of these plants. Higher root dry matter accumulation by hairy vetch and woolly-pod vetch may have contributed to better growth, yield and N₂ fixation of these species.

Nodulation

Nodule formation is a very complex process that readily gets disrupted as a result of effects from environment, the rhizobia themselves and/or genetic determinants (Cooper & Schere, 2012; Theis et al., 1991; Caetano-Anolles & Gresshoff, 1991). The rhizosphere environment strongly affects the symbiotic interaction between rhizobia and their host legumes. Soil factors that influence plant and rhizobial growth such as acidity, temperature, moisture, fertility etc. influence infection and nodulation of legumes (Cooper & Schere, 2012). The range of variation in nodule number, nodule dry matter accumulation and nodulation rate  (Table 5) in response to inoculation of Vicia species suggests that suitable native rhizobia may not be present in the soil (Date, 2000). Moreover, the appearance of few nodules and a wide range of variation between species was also observed by Nutman (1975) who attributed these observations to the proportional differences in the number of infected root hairs, which is host controlled.

Though all the species developed nodules on their roots, the nodules of successfully inoculated legumes were pink to red in color and larger in size than unproductive nodules, most uninoculated plants formed white, smaller and fewer nodules per plant. This phenomenon is in agreement with descriptions of Jennings (2004) and Bulter & Evers (2004) who observed that on legume plants ineffective strains form many small nodules on the legume root but fix little or no nitrogen. The higher nodulation ratings of inoculated legumes showed that, effective nodules were developed on main roots of the plants. Mature and effective (N₂ fixing) nodules are often clustered on the primary root and have pink to beef stick red centers (Jennings, 2004).

Forage Quality

Herbage quality attributes are important in evaluation of forage legumes (Larbi et al., 2010). This attribute is also important for selecting feed legumes for soil fertility enhancement as it influences the amount of soil organic carbon, and the rate and extent at which minerals are released from the herbage into the soil. Improvement in CP content of feed legumes (Table 6) has great nutritional, economic and ecological advantages as these forage legumes are an efficient N source and provide quality feed with a high digestibility (Longo et al., 2012; Havilah, 2011). The importance of forage legumes in livestock (Assefa, 1999) and crop production is no longer questioned. Legumes in general and vetches in particular are excellent sources of N for livestock feed. It is reported that, Vicia species are rich in protein, minerals (Bonsi et al., 1994) and have lower fiber content. Jennings (2004) reported that, well-nodulated legumes contain large amount of protein, calcium, magnesium and other essential elements.

Nitrogen Fixation

Many research reports indicate that hairy vetch (Hartwig & Amnon, 2002; Anugroho et al., 2009a, Seo & Lee, 2008; Bongs & Diamon, 2008; Campiglia et al., 2010) and woolly-pod vetch (Smith & Valenzuela, 2002) are vigorously growing and most efficient nitrogen fixing forage legumes. The obtained result is also in agreement with these findings (Table 7). Differences in phenotype (plant height, vigor, and days to flowering), dry matter accumulation and tolerance to low temperature and acidity might contribute to variations in nitrogen fixation by these species. From field observation (unpublished data) hairy vetch and woolly-pod vetch are tolerant to low temperature which is consistent with literature reports (Campiglia et al., 2010). Low temperature is reported to delay plant development, to reduce dry matter accumulation and to delay nodulation which has a negative effect on N₂ fixation (Robin et al., 2005). This might be the case for low N₂ fixation by common vetch and narbon vetch which are sensitive to low temperature.

The ultimate goal for establishing efficient legume inoculation is to improve and exploit the huge biological N resource that is available naturally. This is particularly important for developing countries like Ethiopia where diverse and complex sets of social, economic and environmental factors are challenging the small-scale farming system. Our results confirmed the specificity of host and Rhizobium interaction and their effect on yield, quality, response to inoculation and N₂ fixation of legumes. As pointed out by other authors (Campiglia et al., 2010; Robin et al., 2005; Chalk, 1991; Peoples and Crasswell, 1992) the most important factors limiting the N₂ fixation ability of legumes are intrinsic to host and bacteria and they also depend on the environment especially the climate. Although the potential benefits of Rhizobium inoculation to legumes has long been known several years before, research and use of this technology in Ethiopia is still very limited, especially on forage legumes. Even if, inoculation success/failure is highly site specific (Singleton et al., 1992), lack of literatures especially on forage legumes makes generalization difficult only based on our findings. However, as an effort to benefit from Rhizobium inoculation and forage legumes, the obtained result offers an attractive alternative to N input and give a useful insight into the significance of forage legumes, needs to improve their potential and utilization.

Conclusion

The present study demonstrates that, Rhizobium inoculation can improve N₂ fixation, growth and quality of forage legumes. Fixation of atmospheric N₂ by nodulated legumes is cost-efficient and an important source of N for food and agricultural production. The higher N yield obtained from woolly-pod vetch and hairy vetch in this study also shows the potential contribution of nitrogen fixing bacteria to improve crop-livestock production systems of the central highland areas of Ethiopia. Thus their inclusion in the farming system offers an opportunity for solving multi-faceted problems of small-scale agriculture in terms of improving soil fertility and providing high quality feed for livestock.

Several suitable forage legumes are available in Ethiopia for potential intercropping, relay cropping and crop rotation which could improve soil fertility, crop yield and roughage quality which in turn will make the system more sustainable. Despite these advantages, the contribution of fodder legumes to the farming system is given so far lower emphasis and their adoption and benefit to the small-scale farmers is minimal. Thus, research on forage legumes improvement and their N₂ fixation inputs should continue as a major interest in the agriculture of developing countries, which often lack N fertilizers and are subject to economic constraints. Being the first attempt to estimate N₂ fixation of forage legumes in Ethiopia, this study can serve as a reference for subsequent works. However, even though the differential determination of estimating the N₂ fixation of legumes is a simple and cheap method it may overestimate the N₂ fixation due to the intrinsic different capabilities of the legume of interest and the reference non-fixing crop to use soil N_2. Therefore, further studies are needed, where other methods (acetylene reduction, ¹⁵N isotope etc.) should be explored in order to establish an efficient and cost effective method for estimating of N₂ fixation by forage legumes. Moreover, this study demonstrates the existence of effective indigenous strains in soils of the Ethiopian highlands which should be further exploited through comprehensive collection, characterization and selection for different potential forage legumes.

 

Acknowledgments

The authors would like to thank the Ethiopian Institute of Agricultural Research (EIAR) for support. Sincere gratitude also goes to the staff members of the Holeta research center of the Forage and Pasture Research, Animal Nutrition and Soil and Water research projects for their support during field and laboratory work.

 

References 

1. Abbeddou S, Rihawi S, Hess H.D, Ińiguez L, Mayer A.C & Kreuzer M. (2011). Nutritional composition of lentil straw, vetch hay, olive leaves and saltbush leaves and their digestibility as measured in fat-tailed sheep. Small Ruminant Research. 96: 126-135.
2. Abd El-Moneim A.M & Cocks P.C. (1993). Adaptation and yield stability of selected lines of Lathyrus spp. under rainfed conditions in west Asia. Euphytica. 66: 89–97.
3. Adjei M.B & Chambeiss C.G. (2002). Nitrogen fixation and inoculation of forage legumes. An electronic publication of agronomy department, University of Florida. p. 1-10. hhtp://edis.ifas.ulf.edu.
4. Allison L.E. (1960). Wet combustion apparatus and producer for organic and inorganic carbon in soil. Soil Science Society of America Proceeding. 24: 36-40.
5. Anugroho F, Kitou M, Nagumo F, Kinjo K & Tokashiki Y. (2009a). Growth, nitrogen fixation and nutrient uptake of hairy vetch as a cover crop in sub-tropical region. Weed Biology and Management. 9: 63-71.
6. AOAC (1990). Official methods of analysis. 15^th edition. Association of Analytical Chemist, (AOAC). Inc., Arlington, Virginia, USA.
7. Argaw A. (2012). Evaluation of symbiotic effectiveness and size of resident R. Leguminosarum var. viciae nodulating Lentil (Lens culinaris Medic.) in some Ethiopian soils. Archives of Agronomy and Soil Science. p. 1-17.
8. Assefa G, Birhanu T, Gizachew L, Dejene M & Geleti D. (2006). Major herbaceous forage legumes: Some achievements in species and varietal evaluation in Ethiopia. In: Ali K, Keneni G, Ahmed S,  Rajendra M, Surendra B, Khaled M & Halila M.H, editors. Food and Feed Legumes of Ethiopia: Progress and prospects. Proceedings of the workshop on food and forage legumes, 22-26 September 2003. Addis Ababa, Ethiopia.
9. Assefa G, Feyissa F, Minta M & Tekletsadik T. (2011). Forage crops productivity and integration with barley in Ethiopia. In: Mulatu B & Grando S, editors. Barley research and development in Ethiopia. Proceedings of the 2^nd national barley research and development review workshop. 28-30 November 2006, Holeta, Ethiopia.
10. Assefa G. (1999). Feed resource assessment and evaluation of forage yield, quality and intake of oats and vetches grown in the highlands of Ethiopia. M.Sc. Thesis. Swedish University of Agricultural Science. Uppsala. p. 1-153.
11. Atemkeng M.N, Muki T.J, Park J & Jifon J. (2011). Integrating molecular tools with conventional breeding strategies for improving phosphorus acquisition by legume crops in acid soils of sub-Saharan Africa. Biotechnology and molecular biology, Review. 6(7): 142-154.
12. Badrzadeh M, Zaragarzadeh F & Esmaielpour B. (2008). Chemical composition of some forage Vicia spp. in Iranian Journal of Food, Agriculture and Environment. 6: 178–180.
13. Bekere W & Hailemariam A. (2012). Influences of inoculation methods and phosphorus levels on nitrogen fixation attributes and yield of Soybean (Glycine max L.) at Haru, Western Ethiopia. American Journal of Plant Nutrition and Fertilization Technology. 2(2): 45-55.
14. Bongs C and Daimon H. (2008). Effect of hairy vetch incorporated as green manure on growth and N uptake of Sorghum crop. Plant Production Science. 11: 211-216.
15. Bonsi M.L.K, Osuji P.O, Nsahlai I.V & Tuah A.K. (1994). Graded levels of Susbania susban and Leucaena leucocephala as supplements to teff (Eragrostis tef  (Zucc.) Trotter) straw given to Ethiopian Menz sheep. Animal Production. 59: 235-241.
16. Bulter T. J & Evers G. (2004). Inoculation, nodulation, nitrogen fixation and transfer. Texas cooperative extension (press).
17. Caetano-Anolles G & Greeshof P.M. (1991). Plant genetic control of nodulation. Annual Review of Microbiology. 45: 345-382.
18. Campiglia E, Caporali F, Radicetti E & Mancinelli R. (2010). Hairy vetch (Vicia villosa Roth.) cover crop residue management for improving weed control and yield in no-tillage tomato (Lycopersicon esculentum Mill.) production. European Journal of Agronomy. 33: 94-102.
19. Chalk P.M. (1991). The contribution of associative and symbiotic nitrogen fixation to the nitrogen nutrition of non-legumes. Plant and Soil. 132: 29-39.
20. Cooper J.E and Schere H.W. (2012). Nitrogen fixation. Plant-soil relationship, part II. p. 389-408.
21. Date R.A. (2000). Inoculated legumes in cropping systems of the tropics. Field crops research. 65: 123-136.
22. Day P.R. (1965). Particle fractionation and particle size analysis. In: Black C.A, editor. Methods of soil analysis, Part I: American Society of Agronomy. p. 545-567.
23. De Jong R, Qian B & Yang J.Y. (2008). Modeling nitrogen leaching in Prince Edward Island under climate change scenarios. Canadian Journal of Soil Science. 88: 61-78.
24. EARO (Ethiopian Agricultural Research Organization) (2000). National forage seed production research strategy. Addis Ababa, Ethiopia. p. 1-60.
25. Fageria N.K & Moreira A. (2011). The role of mineral nutrition on root growth of crop plants. Advances in Agronomy. 110: 251-331.
26. Firincioğlue H.K, Ǜnal S, Erbektas E & Doğruyol L. (2010). Relationships between seed yield and yield components in common vetch (Vicia sativa ssp. sativa) populations sown in spring and autumn in central Turkey. Field Crops Research. 116: 30–37.
27. Foth H.D. (1990). Fundamentals of soil science. John Wiley and Sons, Inc. p. 164-169.
28. Gebrehiwot L & Tadesse A. (1985). Pasture research and development in Ethiopia. In: Kategile J.A, editor. Pasture improvement research in Eastern and Southern Ethiopia. Proceedings of workshop held in Harare, Zimbabwe, 17-21 September 1984. p. 77-91.
29. Getahun M, Assefa G, Tedla A, Janka E & Mengistu A. (2002). Forage and pasture crops genetic resources conservation and research. Stock taking for biodiversity conservation. National strategy and action plan project, Ethiopia. Institute of Biodiversity Conservation and Research (IBCR). Addis Ababa, Ethiopia.
30. Giller K.E.  (2001). Nitrogen fixation in tropical cropping system. 2^nd edn. CABI publication. p. 90-93.
31. Hailemariam A & Tsigie A. (2006). Biological nitrogen fixation research in food legumes in Ethiopia. In: Ali K, Keneni G, Ahmed S, Malhotra R, Beniwal S, Makkouk K & Halila M.H, editors. Food and forage legumes of Ethiopia progresses and prospects. Proceedings of the national workshop on food and forage legumes, September 22-26, 2003, Addis Ababa, Ethiopia.
32. Hardarson G & Danso S.K.A. (1993). Methods for measuring biological nitrogen fixation in grain legumes. Plant and Soil. 152:19-23.
33. Hartwig N.L & Ammon H.U. (2002). Cover crops and living mulches. Weed science. 50: 688-699.
34. Havilah E.J. (2011). Forage and Pasture: Annual forage and pasture crops- establishment and management. Elsevier. p. 563-575.
35. Jennings J. (2004). Forage legume inoculation. In: United States Department of Agriculture and County Governments Cooperating, Agriculture and natural resources. University of Arkansas. http://www.uaex.edu.
36. Landon J.R. (1991). Booker tropical soil manual: a handbook for soil survey and agricultural land evaluation in the tropics and subtropics. Harlow: Longman Scientific & Technical.
37. Larbi A, Abd El-Moneim A.M, Nakkoul H, Jammal B & Hassan S. (2011). Intra-species variation in yield and quality determinants in Vicia species: 3. Common vetch (Vicia sativa ssp. Sativa L.). Animal Feed Science and Technology. 164: 241-251.
38. Larbi A, Abd El-Moneim A.M, Nakkoul H, Jammal B & Hassan S. (2010). Intra-species variations in yield and quality determinants of Vicia species: 2. Narbon vetch (Vicia narbonensis L.). Animal Feed Science and Technology. 162: 20-27.
39. Lira Junior M.A, Lima A.S.T, Arruda J.R.F & Smith D.L. (2005). Effect of root temperature on nodule development of bean, lantil and pea. Soil Biology and Biochemistry. 37: 235-239.
40. Lithaourgidis A.S, Vasilakoglou I.B, Dhima K.V, Dordas C.A & Yiakoulaki M.D. (2006). Forage yield and quality of common vetch mixtures with oat and triticale in two seeding ratios. Field crops research. 99: 106-113.
41. Liu G.D, Wu W.L & Zhang J. (2005a). Regional differentiation of non-point source pollution of agriculture-derived nitrate nitrogen in groundwater in northern china. Agriculture, Ecosystems and Environment. 107: 211-220.
42. Longo C, Hummel J, Liebich J, Bueno I.C.S, Burauel P, Ambrosano E.J, Abdalla1 A.L, Anele U.Y & Sudekum K.H. (2012). Chemical characterization and in vitro biological activity of four tropical legumes, Styzolobium aterrimum, Styzolobium deeringianum, Leucaena leucocephala, and Mimosa caesalpiniaefolia, as compared with a tropical grass, Cynodon spp. for the use in ruminant diets. Czech Journal of Animal Science. 57(6): 255–264.
43. Lopez S, Davies D.R, Giraldez F.J, Dhanoa M.S, Dijkstra J & France J. (2005). Assessment of nutritive value of cereal and legume straws based on chemical composition and in vitro digestibility. Journal of the Science of Food Agriculture. 85: 1550–1557.
44. Mekonnen K. (2007). Evaluation of selected indigenous and exotic tree and shrub species for soil fertility improvement and fodder production in the highland area of western Shewa, Ethiopia. PhD Thesis. University of natural resources and applied life sciences, Vienna.
45. Miller C.E, Turk L.M & Foth H.D. (1965). Fundamentals of Soil Science. 4^th ed. Wiley: New York. p. 65.
46. NifTAL (1979). International Network of Legume Inoculation Trials. NifTAL project, University of Hawaii, College of Tropical Agriculture and Human Resource, USAID. Honolulu.
47. Nnadi L.A & Haque I. (1986). Forage legume-cereal systems: Improvement of soil fertility and agricultural production with special reference to sub-Saharan Africa. In: Haque I, Jutzi S & Neate P.J.H, editors. Potentials of forage legumes in farming systems of sub-Saharan Africa. Proceedings of workshop held at ILCA, Addis Ababa, Ethiopia. 16-19 September 1985. p. 330-345.
48. Nutman P.S. (1975). Rhizobium in the soil. In: Walker N, editor. Soil Microbiology. Buthers Worth, London. p 325-340.
49. Olsen S.R, Cole F.S & Dean L.A. (1965). Estimation of available phosphorus in soils by extraction with sodium carbonate. USDA Cir. No. 939.
50. Pal U.R & Saxena M.C. (1975). Response of soybean to symbiosis and nitrogen fertilization under humid sub-tropical conditions. Experimental Agriculture. 11: 221- 226.
51. Pal U.R. (2004). Crop physiology. Teaching material. Alemaya University, Harar, Ethiopia. p. 108.
52. Peoples M.B & Crasswell E.T. (1992). Biological nitrogen fixation investments, expectations and actual contributions to agriculture. Plant and Soil. 141: 13-30.
53. Peoples M.B, Faizah A.W, Rerkasem B & Herridge D.F. (1989). Methods for evaluating nitrogen fixation by nodulated legumes in the field. ACIAR No. 11. p. 76.
54. Peoples M.B & Herridge D.F. (1990). Nitrogen fixation by legumes in tropical and sub-tropical agriculture. Advances in Agronomy. 44: 155-167.
55. Ranist V.E, Verloo M, Demeyer A & Pauwels J.M. (1999). Manual for soil chemistry and fertility laboratory. p. 50-100.
56. Rao M.R & Mathuva M.N. (2000). Legumes for improving maize yields and income in semi-arid Kenya. Agriculture, Ecosystem and Environment. 78: 123-137.
57. Robert J, Okalebo J.R, Gathua K.W & Paul L.W. (2002). Laboratory method of plant analysis: A working manual. 2^nd. Ed. Nairobi, Kenya. p. 20-70.
58. Robin C, Tubeileh K.S, Obaton M & Guckert A. (2005). Nitrogen fixation and growth of annual Medicago-Sinorhizobium associations at low temperature. European Journal of Agronomy. 22: 267-275.
59. Sainju U. M, Singh B.P & Whitehead W.F. (2005a). Tillage, cover crops, and nitrogen fertilization effects on cotton and sorghum root biomass, carbon, and nitrogen. Agronomy Journal. 97: 1279–1290.
60. Seo J.H & Lee H.J. (2008). Mineral nitrogen effects of hairy vetch (Vicia villosa Roth) on maize (Zea mays L.) by green manure amounts. Agronomy Journal. 7: 272-276.
61. Smith J & Valenzuela H. (2002). Green manure crops: woolly-pod vetch. Cooperative Extension Service. College of Tropical Agriculture and Human Resources. University of Hawai press.
62. Thies J.E, Singleton P.W & Bohlool B.B. (1991). Modeling symbiotic performance of introduced rhizobia in the field by the use of indices of indigenous population size and nitrogen status of the soil. Applied and Environmental Microbiology. 57: 29-37.
63. Turk M, Albayrak S & Yuksel O. (2007). Effects of phosphorus fertilization and harvesting stages on forage yield and quality of narbon vetch. New Zealand Journal of Agricultural Research. 50: 457–462.
64. Somasegaran P & Hoben H.J. (1985). Methods in Legume-Rhizobium Technology. University of Hawaii NifTal Project and MIRCEN. Department of of Agronomy and Soil Science. p. 245-254.
65. Zahran H.H. (2001). Rhizobia from wild legumes: diversity, taxonomy, ecology, nitrogen fixation and biotechnology. Journal of Biotechnology. 91: 143-153.