Indigenous rhizobia population influences the effectiveness of Rhizobium inoculation and need of inorganic N for common bean (Phaseolus vulgaris L.) production in eastern Ethiopia

Abstract

Background

Supplement with inorganic N application is essential to improve the common bean production in sub-Saharan Africa. However, the influence of indigenous rhizobial population on the inorganic N requirement with Rhizobium inoculation to secure sustainable way of common bean production system is not well known. The effect of different rates of N application either alone or in combination with Rhizobium inoculation on the nodulation, yield and yield traits of common bean cultivated in soils with different rhizobial population were conducted.

Methods

Twelve treatments were produced by factorially combined six levels of N fertilizer (0, 20, 40, 60, 80 and 100 kg N ha^sup -1^) and two Rhizobium inoculation treatments (inoculated and uninoculated). The treatments were laid out in randomized completely block design and all treatments were replicated three times.

Result

Regardless of soil types, nodule number and nodule dry weight decreased with increasing rates of N application. 20 kg N ha^sup -1^ both alone and in combination with Rhizobium inoculation resulted in the largest nodulation in all soil types. The largest nodulation were induced in soil with large rhizobial population. Rhizobium inoculation significantly (P < 0.05) improved yield and yield traits of common bean. Moreover, our result revealed that the largest values of investigated traits were observed in inoculated treatment, as compared to the corresponding N rates of uninoculated treatments. The 20, 100 and 40 kg N ha^sup -1^ treatments resulted in significantly greater plant total tissue N at soil types with small, medium and large rhizobial population, respectively, as compared to unfertilized control. The highest total biomass yield (TBY) and grain yield (GY) at soil types with small and medium rhizobial population were obtained by the 100 kg N ha^sup -1^ treatment in combination with Rhizobium inoculation, while 20 and 40 kg N ha^sup -1^ applications produced the greatest TBY and GY, respectively, in soil with large rhizobial population.

Conclusion

These results indicate that N requirement is varied based on rhizobial population and effectiveness of native rhizobia in N2 fixation.

Argaw and Tsigie Chem. Biol. Technol. Agric. (2015) 2:19 DOI 10.1186/s40538-015-0047-z

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Web End = Anteneh Argaw1* and Angaw Tsigie2

Abstract

Background: Supplement with inorganic N application is essential to improve the common bean production in sub-Saharan Africa. However, the inuence of indigenous rhizobial population on the inorganic N requirement with Rhizobium inoculation to secure sustainable way of common bean production system is not well known. The eect of dierent rates of N application either alone or in combination with Rhizobium inoculation on the nodulation, yield and yield traits of common bean cultivated in soils with dierent rhizobial population were conducted.

Methods: Twelve treatments were produced by factorially combined six levels of N fertilizer (0, 20, 40, 60, 80 and 100 kg N ha1) and two Rhizobium inoculation treatments (inoculated and uninoculated). The treatments were laid out in randomized completely block design and all treatments were replicated three times.

Result: Regardless of soil types, nodule number and nodule dry weight decreased with increasing rates of N application. 20 kg N ha1 both alone and in combination with Rhizobium inoculation resulted in the largest nodulationin all soil types. The largest nodulation were induced in soil with large rhizobial population. Rhizobium inoculation signicantly (P < 0.05) improved yield and yield traits of common bean. Moreover, our result revealed that the largest values of investigated traits were observed in inoculated treatment, as compared to the corresponding N rates of uninoculated treatments. The 20, 100 and 40 kg N ha1 treatments resulted in signicantly greater plant total tissueN at soil types with small, medium and large rhizobial population, respectively, as compared to unfertilized control. The highest total biomass yield (TBY) and grain yield (GY) at soil types with small and medium rhizobial population were obtained by the 100 kg N ha1 treatment in combination with Rhizobium inoculation, while 20 and 40 kg N ha1 applications produced the greatest TBY and GY, respectively, in soil with large rhizobial population.

Conclusion: These results indicate that N requirement is varied based on rhizobial population and eectiveness of native rhizobia in N2 xation.

Keywords: Common bean (Phaseolus vulgaris L.), Ethiopia, Indigenous rhizobia, Rhizobium leguminosarum bv.

Phaseoli

*Correspondence: [email protected]

1 College of Agriculture and Environmental Sciences, School of Natural Resources Management and Environmental Sciences, Haramaya University, Dire Dawa, EthiopiaFull list of author information is available at the end of the article

2015 Argaw and Tsigie. This article is distributed under the terms of the Creative Commons Attribution 4.0 International

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Background

Common bean (Phaseolus vulgaris L.) is one of most important food legume cultivated on greater than 4millionha, providing the main source of dietary protein and carbohydrate for eastern and central African peoples, and containing about 2025% of the protein [1]. In Ethiopia, common bean is one of the major grain legumes, with its production centered in small farmers elds where the soil fertility is depleted. Furthermore, the use of nitrogen (N) fertilizer is limited and average yields are low, usually less than 1tonha1 [2]. In contrast, some studies indicated that up to 4600kgha1 seed yield of common bean were obtained from research managed experimental plots in Ethiopia [35]. Low soil fertility, especially low N content, is the most important limiting nutrient status for common bean production in the tropics, including Ethiopia [6, 7].

Due to high price of N mineral fertilizers, their use by subsistent farmers in sub-Saharan Africa (SSA) to increase crop production has been limited. This condition has therefore necessitated an approach to crop production that emphasizes biological N2 xation (BNF).

However, common bean is considered as a poor nitrogen xing plant in comparison to other grain legumes due to its promiscuous nature, i.e., it forms symbiosis with many rhizobia species [8]. On top of this, several ndings revealed that common bean generally responds poorly to Rhizobium inoculation under eld conditions [911]. The reason for the failure to respond inoculation is believed to be due to a high and inefficient population of native common bean nodulating rhizobia in soil [9, 12, 13].

It is indicated that N2 xation in common bean can be increased through highly efficient Rhizobium inoculation [14]. Moreover, elite Rhizobium isolates improved the productivity of common bean, although in soil with high rhizobial nodulating population [1517]. Asad etal. [15] found that the efficient Rhizobium inoculation did not fulll the N needs of common bean. In most cases, common bean is able to x up to 50 kg N ha1 [18], which is less than 50% of the plant N requirement [19]. Therefore, common bean requires mineral N application to achieve substantial yields under the current cropping system in SSA. However, N mineral recommended rates >40kgNha1 suppress nodulation and N2 xation [10, 20, 21]. On the other hand, the application of a small amount of fertilizer N (<30kgNha1) enhanced nodulation, but grain yield improvement was not satisfactory [10, 22]. Higher stimulation of plant growth, N2 xation and grain yield of common bean have been recorded at low levels of N fertilizer applied with Rhizobium inoculation [17, 2325]. Therefore, inoculation trials must emphasize not only the benets of common bean inoculation, but also the combination of that practice with N

fertilization, in order to achieve a decrease in mineral N input, whilst still obtaining maximum yields. However, the amount of N required in conjunction with elite Rhizobium inoculation to get maximum yield of common bean in soil with dierent rhizobial population is unknown. Hence, the objective of this study was to investigate the eect of naturalized common bean rhizobia population on the N rates of applied when alone or in combination with Rhizobium inoculation on nodulation, yield and yield traits of common bean in major growing areas of eastern Ethiopia.

Methods

Study areas

Field experiments were conducted on four locations of Eastern Ethiopia having dierent indigenous rhizobia nodulating common bean in 2012 cropping season. The experimental sites were located in the Hirna [N0913.157 and E04106.488 at an altitude of 1808 m above sea level (m.a.s.l.)], Fedis (N0906.941 and E04204.835 at an altitude of 1669 m.a.s.l.) Babillae (N0913.234 and E04219.407 1669m.a.s.l.) and Hara-maya (N0924.954 and E04202.037 at an altitude of 2020m.a.s.l.) agricultural research centers. The soils had not been inoculated before with rhizobia isolates nodulating common bean. Before sowing, soil samples were taken from 020 cm depth to determine baseline soil properties. Soil samples were air-dried, crushed, and passed through a 2-mm sieve prior to physical and chemical analysis. Details of physical and chemical characteristics of the soil of experimental sites are given in Table1.

Sources ofseeds andRhizobium strain

A common bean var. Dursitu was supplied by Lowland Pulses Research Project, Haramaya University, Ethiopia. Variety was selected based on their yield, their maturity time, and its better performance in eastern Ethiopia. Strain of Rhizobium leguminosarum bv. Phaseoli (HUPvR-16) was obtained from Biofertilizer Research and Production Project, Haramaya University (Hara-maya, Ethiopia). This strain was selected because it was previously found efficient while tested in this region on two improved varieties of common bean under laboratory and greenhouse conditions [26].

Inoculums preparation

Agar slope of HUPvR-16 strain was obtained from Soil Microbiology Research Laboratory, Haramaya University, Ethiopia. For purication, this isolate was preliminarily cultured in YEMA medium (10 g mannitol, 1 g yeast-extract, 1g KH2P04, 0.1g NaCl, and 0.2g MgSO47H2O per liter, pH 6.8) and incubated at 28C for 5days. The pure colony of the isolate was later transferred to YEM broth

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Table 1 Soil analysis ofexperimental sites beforesowing

Soil properties Hirna soil Babillae soil Haramaya soil Fedis soil

pH in H2O 7.25 6.66 7.84 7.76 EC (mS/cm) 0.06 0.04 0.14 0.06

Organic carbon (%) 1.65 0.56 1.96 1.32 Total nitrogen (%) 0.16 0.06 0.12 0.12 Available P (mg kg1) 27.11 2.22 9.94 1.78 Ca (cmol(+)kg1) 39.88 4.18 31 23.12

Mg (cmol(+)kg1) 9.00 3.5 8.7 12.87

Na (cmol(+)kg1) 0.14 0.15 0.33 0.12

K (cmol(+)kg1) 0.80 0.34 0.14 1.09

CEC (cmol(+)kg1) 40.03 6.59 25.98 32.22

Zn (mg kg1) 0.95 0.26 0.11 0.10 B (mg kg1) 0.83 ND 0.15 0.75 NH4-N (mg kg1) 33.77 25.57 20.10

NO3-N (mg kg1) 33.74 27.98 27.75 Clay (g kg1) 49 18 33 36

Silt (g kg1) 39 6 18 45Sand (g kg1) 12 79 49 19Textural class Clay Sandy loam Sandy clay loam Silty clay loam Number of indigenous rhizobia of common bean g1 soil 1.1 104 <10 2.8 103 2.5 102

medium and incubated at 28 C for 5 days with gentle shaking at 120rpm in shaker incubator. By this procedure, cell density in the culture was estimated by measuring optical density (540nm) to determine whether the Rhizobium culture reached the middle or late logarithmic phase. Rhizobium inoculant was prepared by mixing 30g of sterilized decomposed lter-mud with 15ml of broth culture containing HUPvR-16 strain in polyethylene bags. After incubating the inoculated lter-mud for 2weeks at 28C, the count of the Rhizobium was reached 1 109 g1 of inoculant. Populations of rhizobia in the inoculants were determined by duplicate plate counts (Vincent, 1970).

Enumeration ofindigenous rhizobia nodulating common bean

The initial indigenous rhizobia population was determined by the plant infection technique, using inoculation of serially diluted soil on germinated common bean seedling for nodulation assessment following the method of Brockwell etal. [27]. This experiment was conducted under controlled condition in growth chamber. The most probable number (MPN) was calculated from the most likely number, using the MPN tables of Vincent [28]. The rhizobial population that nodulated common bean in all study sites are indicated in Table1.

Experimental design

Field trials on three soil types which had dierent rhizobial population nodulating common bean were

conducted in order to investigate the eect of indigenous rhizobial population on the eect of N rates when applied alone or in combination with Rhizobium inoculation. The treatment eects were evaluated via determination of nodulation, yield and yield traits of common bean. The experimental design was a split plot in randomized complete block design (RCBD) with three replications. Main plot treatments consisted of six levels of inorganic N: 0, 20, 40, 60, 80 and 100kgNha1. Two Rhizobium inoculations (inoculated and uninoculated) were assigned as subplot treatments. Nitrogen fertilizer in each level was divided into two equal parts: (1) the rst part of the N (20kgN ha1) was applied along the furrow by hand and incorporated before planting time, and (2) the remaining parts were applied at owering stages (R3-stage).

The area was moldboard-plowed and disked before planting. The sizes of the main and subplot were 35m2

and 32m2, respectively. There were ve rows per sub-plot and the spacing was 40 cm between rows, 10 cm between plants, 1m between subplots and 1.5 between main plots. Disinfected seeds of common bean were sown after they were moistened with a 20% solution of sucrose and then inoculated (7g inoculant per kg seed) with Rhizobium. Inoculated seeds were hand planted on July 7, 2012. Phosphorus (P) was uniformly applied at planting at rate of 20 kg P ha1 as triple superphosphate. Two seeds were sown per hill. After germination, the plants were thinned to one seedling per hill to obtain about 30 plants per row.

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Weeds were controlled over the growth period with hand hoeing. At late owering and early pod setting stage (R3 stage), ve plants from central rows were randomly chosen and harvested to record number of nodule plant1 (NN), nodule dry weight plant1 (NDW) and shoot dry weight plant1 (SDW). Shoots of the plants were dried and later ground to pass a 0.5cm sieve. Total N determinations were done by the Kjeldahl method of Bremner [29]. At physiological maturity stage on October 30, 2012, yield and yield traits of common bean were recorded. Number of pods plant1 (NPP), number of seeds plant1 (NSP), 100 seed weight, total biomass yield (TBY) and grain yield (at 13 % moisture content) (GY) were determined.

Data analysis

Data were submitted to analysis of variance using SAS version 9.1. Statistically signicant dierences between means were also determined by the LSD test. The bar graphs were constructed using Microsoft excel version 10.

Result anddiscussion

Nodulation

The soil samples from four experimental sites were aseptically collected to enumerate the rhizobia population

that nodulated common bean by using the plant infection method. The result of this experiment revealed that the rhizobia of common bean varied from <10 to 105g1 of soil. Based on this rhizobial population, the experimental sites were grouped into three soil types. Accordingly, Babillae soil had <100 rhizobia of common bean g1 soil.Rhizobia population >1000g1 of soil was found in Hara-maya and Hirna soils. While the rhizobial population in Fedis soil was between 100 and 1000g1 soil. Therefore, Babillae, Fedis, and Haramaya and Hirna soils were categorized into low, medium and high rhizobia containing soil types, as it has been previously described by Howieson and Ballard [30]. None of common bean cultivating history in Babillae and Fedis sites could be attributed to lower rhizobial population nodulating common bean [31]. Continuous cultivation of the host plant could increase rhizobial population at Haramaya and Hirna soils [32].

Dierent rates of N application either alone or in combination with Rhizobium leguminosarum bv. Phaseoli inoculation signicantly (P < 0.05) aected common bean nodulation in all study sites (Table 2). In all soil types, the NN and NDW were signicantly decreased with increasing rates of N application either alone or in combination with inoculation. Other authors have found similar trends of nodulation along N rates with dierent

Table 2 Nodulation status and shoot dry weight of common bean var. Dursitu along dierent rates of N application withand withoutinoculation ofRhizobium leguminosarum bv. Phaseoli atselected areas ofeastern Ethiopia

Treatments NN NDW SDW

Soil type 1 Soil type 2 Soil type 3 Soil type 1 Soil type 2 Soil type 3 Soil type 1 Soil type 2 Soil type 3

Control 28.33d 71.67bcd 163.00abcd 0.1217cd 0.1920bc 0.4407b 36.23cd 31.37e 49.92b 20 kg N ha1 88.67c 146.67a 206.00a 0.1880b 0.4097a 0.3399bc 35.30cd 38.03cde 58.13ab 40 kg N ha1 65.00c 106.67b 149.67abcde 0.1263cd 0.3963a 0.2299bc 41.17bc 48.57bcd 60.77ab 60 kg N ha1 18.67d 45.00def 91.33cde 0.0437d 0.1727bc 0.1300c 49.97ab 53.23abc 63.60ab 80 kg N ha1 31.00d 45.00def 93.00cde 0.1033cd 0.1610bcd 0.1621c 47.53ab 55.70ab 63.67ab 100 kg N ha1 22.33d 20.00f 68.00e 0.0193d 0.0160e 0.2127bc 44.83abc 56.33ab 76.47a Rhizobium sp. 82.33c 90.67bc 186.67ab 0.3063ab 0.2533b 0.7184a 30.77d 39.00cde 59.07ab Rhizobium sp. + 20 kg N ha1 161.67a 64.33cde 178.67abc 0.3900a 0.2010bc 0.7111a 42.63bc 36.40de 70.50ab

Rhizobium sp. + 40 kg N ha1 124.33b 46.67def 111.50bcde 0.3207ab 0.0897cde 0.3337bc 52.93a 64.90a 78.03a

Rhizobium sp. + 60 kg N ha1 26.00d 27.33ef 71.50de 0.1530cd 0.0516de 0.1596c 47.47ab 52.83abc 75.68a

Rhizobium sp. + 80 kg N ha1 17.33d 20.00f 170.00abc 0.0287d 0.0340e 0.3238bc 50.77ab 51.70abcd 76.38a

Rhizobium sp. + 100 kg N ha1 19.67d 22.33f 63.17e 0.0210d 0.0279e 0.2124bc 43.93abc 51.97abcd 66.92ab

Mean 57.11 58.86 129.38 0.1518 0.1671 0.3312 43.63 48.35 66.59 F value 66.17*** 29.26*** 7.05*** 21.55*** 33.36*** 12.78*** 11.46*** 9.80*** 3.71** LSD 30.06 37.15 93.59 0.1389 0.119 0.27 10.22 16.20 22.32 CV (%) 17.88 21.44 36.85 31.07 24.19 41.54 7.95 11.38 17.07

Means in the same column followed by the same letter are not signicantly dierent at the 5% probability level by Tukeys test NS non-signicant, NN nodule number, NDW nodule dry weight, SDW shoot dry weight* Signicant at 0.05; ** signicant at 0.01; *** signicant at 0.001

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legume pulses [21, 33, 34]. In soil with low rhizobial population, Rhizobium inoculation alone produced signicantly higher NN and NDW than the uninoculated treatments. In contrast, the data indicated the non-signicant increases of NN and NDW due to inoculation in soil types with medium and high rhizobial population. Similarly, the eect of inoculation on nodulation of common bean varied due to dierent indigenous rhizobial population, as previously observed by Asad etal. [15]. In soil having low rhizobial population, Rhizobium inoculated with 20 and 40kgNha1 resulted in signicantly higher NN than the remaining N treatments. Moreover, Rhizobium inoculated with 20 kg N ha1 produced the highest NDW. The overall eect of inoculation on NN and NDW in this soil type was higher than for the corresponding N rates of applied without inoculation (Fig.1a, b), thus showing the competitive advantage of inoculated Rhizobium over the indigenous rhizobial population. Similarly, low rate of N with inoculation in soil with low indigenous rhizobial population resulted in an enhancement of nodulation [24]. In contrast, N application as low as 15kgNha1 and applied at sowing, suppressed nodule dry weight of common bean [25]. Furthermore, N applied at planting had benecial eect on nodulation at late growth stage [35].

The soil type with medium rhizobial population responded to the 20kgNha1 with signicantly higher NN and NDW than the other treatments, followed in the order by that obtained from 40kgNha1. On top of this, it was also observed a slight reduction of NN and NDW due to inoculation as compared to the uninoculated treatment (Fig.1a, b). Similar result was previously observed by Msumali and Kipe-Nolt, [36], when indigenous rhizobia may have been more potent than inoculated Rhizobium strain. On the other hand, for soil having high rhizobial population, the 20 kg N ha1 treatment alone gave the highest NN, although without signicant dierence from that obtained with either Rhizobium inoculation alone or 40 kg N ha1 alone or inoculation applied with 20kgNha1. Moreover, the highest NDW were obtained from Rhizobium inoculation applied either alone or in combination with 20kgNha1. In general, it was observed a slight increment of NN and a remarkable improvement of NDW due to inoculation, as compared to the uninoculated treatments with corresponding rates of N (Fig. 1a, b). Similar to this nding, a signicant improvement of nodulation due to inoculation in soil with >1000 rhizobial population was observed by Hungria etal. [17]. Inoculation together with a 20kgNha1 treatment resulted in the highest NDW at soil with low and high rhizobial population. While the 20 kg N ha1 treatment alone gave the highest NDW in soil with medium rhizobial population. The highest NN produced

in soil with low, medium and high rhizobial population were 161.67, 146.67 and 206.00, respectively. These various NN in dierent soil types could be attributed to different number of rhizobia present in these three soil types. Similarly, Patrick and Lowther [37] found that the size of rhizobial population aects nodulation. Previous work conrmed that the largest the soil native rhizobial population, the greater is the nodulation [38]. The lowest NN and NDW produced in soil having relatively lower rhizobial population was previously observed by Cheminingwa and Vessey [21].

Shoot dry weight

The experimental treatments (dierent rates of N application solely and in combination with Rhizobium inoculation) resulted in signicant variation of SDW (Table2). In all soil types, a signicant improvement of SDW was observed with increasing rates of N applied either alone or in combination with Rhizobium inoculation. In soil with low and medium rhizobial population, Rhizobium inoculation together with the 40kgNha1 treatment, in which the highest nodulation was produced, resulted in signicantly higher SDW than those produced at N rates lower than 40kgNha1 in both inoculation treatments. This indicates the importance of higher nodulation for SDW production of common bean. In contrast, the high N rate application decreased nodulation, but an increase of above ground biomass production was observed [21]. In soil having high rhizobial population, the non-significant dierence in SDW among N treatments, excluding the control, was observed. This implies that the sole N reserves in soil with lower N rate of application could have been sufficient for maximum shoot biomass production of common bean at R3 stage. The highest SDW obtained from soils having low, medium and high rhizobial population were 52.93, 64.90 and 78.03 g, respectively. All these shoot biomass were obtained from Rhizobium inoculated with 40 kg N ha1. It has been shown that number of native rhizobia had a detrimental impact on productivity of above ground dry biomass [39]. Denton et al. [40] found increased shoot biomass production with increased Rhizobium inoculation rate. In all soil types, the lowest SDW were produced for the control treatments (uninoculated and unfertilized). This suggests that the N is the major limiting nutrient for common bean production in all study sites.

Number ofpods perplant

The treatments of this experiment aected signicantly (P < 0.05) the NPP in all soil types (Table 3). In soil having low rhizobial population, 20kgNha1 alone gave the highest NPP. Inoculation with 60 kg N ha1 resulted in signicantly higher NPP than those produced

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a

c

Soil type 1 Soil type 2 Soil type 3

Soil type 1 Soil type 2 Soil type 3

9000

160

8000

140

7000

TBY(kg/ha)

120

6000

100

NN

5000

80

4000

60

3000

40

2000

20

1000

0

0

uninoculated Inoculated

uninoculated Inoculated

Inoculation treatments

Inoculation treatments

b

d

Soil type 1 Soil type 2 Soil type 3 Soil type 1 Soil type 2 Soil type 3

3000

0.5

2500

NDW (g/plant)

0.4

2000

0.3

GY(kg/ha)

1500

0.2

1000

0.1

500

0

uninoculated Inoculated

0

uninoculated Inoculated

Inoculation treatments

Inoculation treatments

e

Soil type 1 Soil type 2 Soil type 3

6

5

4

PTTN (%)

3

2

1

0

uninoculated Inoculated

Inoculation treatments

Fig. 1 The eect of indigenous rhizobial population on the eectiveness of inoculation on a nodule number (NN), b nodule dry weight (NDW), c grain yield (GY), d total biomass yield (TBY), e plant total tissue N (PTTN). Soil type 1soil having <100 rhizobial population, soil type 2soil having rhizobial population between 100 and 1000 and soil type 3soil having rhizobial population >1000

for the control and inoculation alone, in soils having medium and high rhizobial population. Similarly, a signicant improvement of NPP ranging from 20.2 at the

control to 24.15 at N treated plants was obtained from faba bean [41]. The highest NPP produced in soils having low, medium and high were 18.63, 24.99 and 31.05,

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Table 3 Number ofpods perplant, number ofseeds perpod and100 seed weight ofcommon bean var. Dursitu alongdifferent rates ofN application withand withoutinoculation ofRhizobium leguminosarum bv. Phaseoli atselected areas ofeastern Ethiopia

Treatments NPP NSP 100 seed weight

Soil type 1 Soil type 2 Soil type 3 Soil type 1 Soil type 2 Soil type 3 Soil type 1 Soil type 2 Soil type 3

Control 12.55bc 10.33d 20.89c 4.61b 5.40bc 5.82b 19.77c 20.90cd 19.33a 20 kg N ha1 18.63d 18.66ab 20.83c 6.78a 6.07abc 6.62ab 21.30ab 22.80abc 19.18a 40 kg N ha1 17.11ab 23.22ab 23.00bc 6.55a 6.30ab 6.70ab 21.37ab 22.07bc 18.92a 60 kg N ha1 16.00abc 18.00abc 25.22abc 6.44a 6.97a 6.92ab 21.17abc 22.13abc 19.42a 80 kg N ha1 14.89abc 22.00ab 25.83abc 6.99a 6.40ab 7.13a 21.20abc 22.90ab 19.42a 100 kg N ha1 16.44ab 22.22ab 25.78abc 6.66a 6.63a 6.70ab 21.57a 22.90ab 19.02a Rhizobium sp. 10.78c 11.22 cd 23.66bc 5.33ab 5.50bc 6.50ab 20.00bc 19.90d 19.28a Rhizobium sp. + 20 kg N ha1 13.66abc 16.44bcd 29.33ab 6.89a 5.20c 6.65ab 21.07abc 22.57abc 18.87a

Rhizobium sp. + 40 kg N ha1 16.00abc 22.00ab 29.11ab 6.66a 6.30ab 6.85ab 21.40ab 23.03ab 18.97a

Rhizobium sp. + 60 kg N ha1 16.22abc 24.99a 31.05a 6.22ab 6.30ab 6.35ab 22.17a 22.20abc 18.83a

Rhizobium sp. + 80 kg N ha1 16.44ab 22.11ab 28.89ab 6.99a 6.40ab 6.72ab 21.47ab 21.37bcd 18.87a

Rhizobium sp. + 100 kg N ha1 15.22abc 22.22ab 27.05abc 6.89a 6.00abc 6.38ab 20.97abc 24.07a 19.10a

Mean 15.33 19.45 25.89 6.42 6.12 6.61 21.12 22.24 19.10F value 3.93** 11.06*** 5.86*** 4.68*** 6.87*** 2.11* 5.09*** 8.41*** 0.21 ns LSD 5.48 7.20 6.66 1.72 1.02 1.10 1.49 1.94 2.30 CV (%) 12.13 12.58 13.10 9.11 5.64 8.48 2.38 2.96 6.12

Means in the same column followed by the same letter are not signicantly dierent at the 5% probability level by Tukeys test NS non-signicant, NPP number of pods per plant, NSP number of seeds per pod* Signicant at 0.05; ** signicant at 0.01; *** signicant at 0.001

respectively, conrming the positive eect of indigenous rhizobial population on NPP.

Number ofseeds perpod

The data revealed signicant variation of NSP due to the treatments (Table3). In all soil types, NSP increased with increasing rates of N application solely and in combination with Rhizobium inoculation. Previous study reported that N nutrition increased the seeds per pod [42, 43]. In soil having low rhizobial population, signicantly higher NSP (6.99) was recorded at 80 kg N ha1 and inoculation in conjunction with 80 kg N ha1 as compared to the control. In soils having medium rhizobial population, 60 kg N ha1 resulted in signicantly higher NSP than those produced at N rates below 20 kg N ha1 in both inoculation treatments. 80 kg N ha1 resulted in the highest NSP in soil having high rhizobial population. Non-signicant dierences in NSP obtained from different N rates, excluding control were observed for both inoculation treatments, in soils with low and high rhizobial population.

100 seed weight

The 100 seed weight of common bean was signicantly (P < 0.05) varied due to the treatments, except those observed in soil having high rhizobial population, in

which this trait exhibited a non-signicant dierence (Table 3). The non-signicant dierence in common bean seed size was previously observed by Mulas et al. [44]. Similar to this, 100 seed weight of soybean was not improved by either inoculation or N fertilizer application [45]. In soil having low rhizobial population, signicantly higher 100 seed weight was obtained from 100kgNha1 and Rhizobium inoculated with 60 kg N ha1, as compared to the control treatment. The sole 100kgNha1 addition and in combination with Rhizobium inoculation gave signicantly higher 100 seed weight compared to the control, in soil having medium rhizobial population. This result is in agreement with that obtained previously by El Hardi and Elsheikh [46] who found that inoculation and N application signicantly improved 100 seed weight of chickpea over the control. N application increased 100 seed weight by 7.4% over those produced for the uninoculated treatment [41].

Total biomass yield

The signicant variation of TBY due to treatments was observed at P 0.05 (Table 4). In all soil types, Rhizobium inoculated with 100kgNha1 resulted in signicantly higher TBY than those produced in the control treatment and inoculation alone. This result supports the ndings of Mulas et al. [44] that inoculation and

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Table 4 Total biomass yield, grain yield andtotal plant tissue N ofcommon bean var. Dursitu alongdierent rates ofN application withand withoutinoculation ofRhizobium leguminosarum bv. Phaseoli atselected areas ofeastern Ethiopia

Treatments TBY GY PTTN

Soil type 1 Soil type 2 Soil type 3 Soil type 1 Soil type 2 Soil type 3 Soil type 1 Soil type 2 Soil type 3

Control 2055.6ef 2077.8c 4917.6c 1025.65f 1082.13cd 1946.05bc 2.3800de 2.5200c 4.0983ab 20 kg N ha1 2277.8def 4018.5ab 5609.5bc 1254.35e 1270.19bc 2121.48abc 3.2433a 3.5533b 4.5300ab 40 kg N ha1 2631.5cd 4333.3a 6351.4abc 1568.52c 1307.13bc 2262.27ab 2.8533abcd 3.7800ab 4.6533a 60 kg N ha1 3131.5abc 4055.6a 6481.5abc 1727.63bc 1289.44bc 2207.04abc 2.5567bcde 3.6933ab 4.0767ab 80 kg N ha1 3082.4bc 4388.9a 6721.8ab 1674.26bc 1280.46bc 2380.60a 2.5167cde 3.8600ab 4.2900ab 100 kg N ha1 3260.8ab 4374.1a 6742.8ab 1830.09b 1454.26ab 2416.44a 3.1567abc 4.2000a 4.1200ab Rhizobium sp. 1787.0f 3133.3b 5472.6bc 996.39f 1005.37d 1941.67c 2.1600e 3.3833b 4.0517ab Rhizobium sp. + 20 kg N ha1 2148.1def 4087.0a 7405.6a 1302.22de 1258.70bc 2169.17abc 3.1800ab 3.3633b 4.1867ab

Rhizobium sp. + 40 kg N ha1 2498.1de 4249.2a 6668.1ab 1211.48ef 1569.72a 2381.16a 2.6567abcde 3.6800ab 4.0800ab

Rhizobium sp. + 60 kg N ha1 3375.4ab 4277.8a 6851.6ab 1697.59bc 1461.85ab 2366.06a 2.7000abcde 3.8200ab 4.0033ab

Rhizobium sp. + 80 kg N ha1 3421.4ab 4462.2a 6486.1abc 1511.57cd 1571.57a 2240.34abc 3.2000ab 3.9200ab 3.9467ab

Rhizobium sp. + 100 kg N ha1 3648.1a 4740.7a 7222.2a 2089.54a 1653.89a 2329.40a 3.0133abcd 3.8767ab 3.7583b

Mean 2776.48 4016.54 6410.89 1490.78 1350.39 2230.14 2.8014 3.6375 4.1496 F value 32.12*** 16.30*** 4.95*** 57.11*** 19.75*** 5.99*** 8.10*** 14.64*** 1.73 ns LSD 557.86 913.75 15,380.6 226.81 226.53 318.67 0.6467 0.5621 0.8943 CV (%) 6.82 7.73 12.56 5.17 5.70 7.28 7.84 5.25 10.98

Means in the same column followed by the same letter are not signicantly dierent at the 5% probability level by Tukeys test NS non-signicant, TBY total biomass yield, GY grain yield, PTTN plant total tissue N* Signicant at 0.05; ** signicant at 0.01; *** signicant at 0.001

inorganic N application enhanced the common bean production in all soil type regardless of the indigenous rhizobial population. Signicant enhancement of above ground biomass production by 22 % due to Rhizobium inoculated with inorganic N over the uninoculated treatment was also observed by Cheminingwa and Vessey [21]. In soil having medium rhizobial population, it was observed a non-signicant dierence in TBY produced at 20kgNha1 and beyond rates of N, with both inoculation treatments. This could have conrmed the presence of eective common beanrhizobia symbiosis at low inorganic N [47] and thus satisfy the N need for boost the biomass production. The control treatment, inoculation alone, and 20 kg N ha1 alone gave signicantly lower TBY than those produced in other treatments in soil having high rhizobial population. In soil having low rhizobial population, statistically lower TBY was produced for control treatment, and for those with 20 and 40kgNha1 either alone or in combination with Rhizobium inoculation, as compared to those produced in other treatments. This may indicate that the native rhizobia in this site are capable but not eective to x N2 to satisfy the

N requirement of common bean. The presence of higher rhizobial population is not an indicator of the symbiotic eectiveness between rhizobia and N derived from the atmosphere [31]. In all soil types, the present study revealed that inoculation slightly increased the TBY as

compared to those obtained from the corresponding N treatments without Rhizobium inoculation (Fig. 1c). The highest TBY produced in soils having low, medium and high rhizobial population were 3648.1, 4740.7 and 7222.2kgha1, respectively. The highest biomass in soil having high rhizobial population was previously conrmed by Furseth et al. [48]. These authors found that yield of soybean positively correlated with soil indigenous rhizobial population across environments.

Grain yield

The GY of common bean revealed signicant variation due to treatments at P 0.05 (Table 4). In soil having low rhizobial population, increasing rates of N application increased GY production, though the highest nodulation and PTTN were recorded at 20 kg N ha1. The 100kgNha1 addition in conjunction with inoculation also gave signicantly higher GY than those produced in other treatments. Similarly, inhibition eect of higher N application on nodulation and nitrogenase enzyme without aecting grain yield was previously determined by Rai [23]. Our result may be conrm that the previous ndings, although N mineral reduced the nodulation and N2 xation and yields of common bean can be improved by increasing N availability [49]. da Silveira et al. [47] also found that common bean responded well up to 200kgNha1. Asad etal. [15] observed that nodulation

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improvement due to inoculation had not signicantly enhanced plant biomass production. This present study also conrms that the common beanrhizobia symbiosis was not satisfactory to fulll the N needs as it has been previously observed by Fesonko et al. [50]. Contrary to this, Denton etal. [40] showed that improvement of shoot N increased the grain yield of faba bean by 1Mgha1 in soil having low rhizobial population. This indicates that biological N2 xation alone is not a sufficient N requirement of common bean to get the local attainable yield in the prevailing environmental condition.

Inorganic N at 100kgNha1 with Rhizobium inoculation gave the highest GY at soil having medium rhizobial population as it has been observed in PTTN. This may indicate that N derived from indigenous and inoculated rhizobia alone did not satisfy the N requirement of common bean. Voisin etal. [51] observed that high N requirement at the seed setting stage was supported by both N2 xation and mineral N supply. Although N fertilizer enhanced the plant N uptake, the common bean production did not improve [25]. The present study indicates a non-signicant dierence in GY obtained from inoculation when either coupled to 40, 60, 80 and 100kgNha1 treatment, or sole application of 100kgNha1 addition. This may indicate the inoculated Rhizobium satises the N requirement of common bean with relatively low inorganic N application.

In soil having high rhizobial population, the 40 kg N ha1 applied in conjunction with Rhizobium resulted in signicantly higher GY than those produced at the control and inoculation alone. Similarly, a previous nding recommended N application to maximize common bean yield in soil having high indigenous rhizobial population [31]. In this soil type, it was recorded a non-signicant dierence in GY at dierent N rates of application, excluding control. Besides this, treatment with low N rate applied together with both inoculation treatments, produced statistically similar GY with those found at higher N rates application. The synergetic eect between low rates of N application and Rhizobium inoculation on common bean production in soil having rhizobial population >1000 was previously conrmed by Hungria et al. [17]. These authors found the highest seed yield from treatments with Rhizobium inoculated together with 15kgNha1 applied at planting and further 15kgNha1 addition at early owering stages. Due to the fact that biological N2 xation is not active at early stage of common bean, a starter dose of N application is required to enhance plant growth and eventually improve the grain yield production [35, 52, 53]. Nitrogen application in addition to starter N reduced nodulation and failed to increase the common bean yield [24]. Vargas etal. [31] found that increasing rates of N decreased

the number of nodules from inoculated cells, whereas it increased that by indigenous rhizobia.

The overall eect of inoculation on GY was slightly decreased in soil having low rhizobial population but slightly improved in soil having medium and high rhizobial population, as compared to that obtained at corresponding rates of N application without inoculation (Fig. 1d). This result supports the nding of Hungria et al. [54] who showed that inoculation increased the yield of common bean as compared to the control treatment. A previous study also reported that an inoculation response was observed in soil having rhizobial population between 300 and 1000 g1 soil [55, 56]. The highest GY produced for soil types having low, medium and high rhizobial population were 2089.54, 1653.89 and 2381.16kgha1, respectively. These GY are comparable with those previously produced at 100kgNha1 N, for in which 2000kgha1 was reported [47]. At least 29% faba bean yield advantage was provided for soil having higher rhizobial population over that yield obtained from low rhizobial population, as determined by Sorwle and Mytton [41].

Plant total tissue N

The PTTN was signicantly varied due to dierent treatments at P0.05 (Table4). In soil having low and high rhizobial population, N application beyond 20kgNha1

with both inoculation treatments resulted in a slight decrease of PTTN. 20kgNha1 application resulted in the highest PTTN in soil having low rhizobial population. The lower PTTN at higher N application could be attributed to the negative eect of inorganic N on xed N through the inhibition of the nitrogenase activity [20, 57].

In soil having medium rhizobial population, progressively larger rates of N application increased the PTTN, though the highest NN and NDW was produced at 40kgNha1. The 100kgNha1 treatment gave signicantly higher PTTN than those obtained from either the control treatment or that with 20kgNha1 applied alone or in combination with Rhizobium inoculation. This could be due to low eectiveness of indigenous rhizobia of common bean in N2 xation. Asad etal. [15] found the signicant improvement of plant total N accumulation of common bean was due to N application rather than to inoculation. This was attributed to the fact that common bean rarely derives more than 50% of its N from symbiotic N2 xation [58].

In soil type having high rhizobial population, 40 kg N ha1 alone resulted in signicantly higher PTTN than that obtained from inoculation with the 100 kg N ha1 addition. Shutsrirung et al. [59] demonstrated that inoculation may not be eective in soil having high eective native rhizobial population. In top of

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this, PTTN was also decreased with increasing rates of N application following both inoculation treatments. This indicates that the native rhizobia in this site might have been more eective in N2 xation than the inoculated stain. Furthermore, inoculation slightly increased the PTTN in soil having low and medium rhizobial population whereas it showed a decreasing trend in soil with high rhizobial population, in comparison to PTTN found for corresponding N rates without inoculation (Fig.1e). The highest PTTN was observed at low, medium and high rhizobial population and reached 3.24, 4.20 and 4.65%, respectively. The highest PTTN could be attributed to a higher nodulation induced by the high rhizobial population and, thus, an enhanced N2 xation [38, 48]. Due to synergetic eect of a starter N application on symbiotic N2 xation, the highest accumulated N in plants was recorded for the 20 kgNha1 application in soil having high rhizobial population [24].

Correlation analysis

The signicant correlation among investigated traits of common bean was observed in all soil types at P<0.05 (Tables 5, 6, 7). The amount of NN produced at soil with low rhizobial nodulating population inversely correlated with TBY (r=0.6754; P0.05) and with GY (r = 0.5629; P 0.05) (Table 5). In this soil type, a negative and strong correlation between NDW with TBY (r=0.7563; P0.01), GY (r=0.6953; P0.05) was observed. NN had an inverse and strong correlation with both SDW (r=0.5958; P0.05) and GY (r=0.6090;

P 0.05) in soil with medium rhizobial population (Table6). Conversely, NDW and GY revealed an inverse and signicant correlation (r=0.6356; P0.05). In soil with high rhizobial population, the correlation analyses

between the NN and both TBY (r = 0.5599; P 0.05) and GY (r = 0.7538; P 0.01) were positive (Table 7), whereas NDW had an inverse and signicant correlation with GY (r = 0.6917; P 0.05). In general, this nding indicates that N mineral fertilizer rather than symbiotic N2 xation had a determinant eect on common bean yield. This result supports the results of the other authors [60], in which NDW and N xation showed negative correlation with yield related parameters. La Favre and Eaglesham [61] also observed an inverse and signi-cant relationship between the starting N application and both NN and NDW. In contrast, Aggarwal [62], who conducted experiment in Malawi, found positive and signi-cant correlations between NN and GY for common bean under inorganic N treatment. Mothapo et al. [38] also reported that plant biomass production correlated positively with NDW when inoculation alone was applied.

In all soil types, also SDW of common bean had a signicant correlation with NPP, NSP, 100 seed weight, TBY and GY. However, NPP and NSP had the non-signicant association with SDW with low and high rhizobial population (Tables5, 6, 7). In particular, in soil with low rhizobial population, all listed growth traits had positive and signicant correlation with SDW at P 0.05 (Table 5).

SDW had also a positive and signicant (P0.05) correlation with 100 seed weight (r = 0.5087) and TBY (r = 0.6996), and even highly signicant (P 0.01)

with NPP (r = 0.7719), NSP (r = 0.7751) and GY (r=0.7270). In the same soil, a positive and strong correlation was also observed between SDW and either NPP (r=0.8726; P0.001), or TBY (r=7378; P0.01) or

GY (r = 0.7496; P 0.01). Highly signicant relationships among SDW, shoot N content and seed yield are previously shown by Pereira and Bliss [63].

Table 5 Correlation amongthe investigated traits ofcommon bean treated dierent rates ofN withand withoutinoculation insoil having rhizobial population nodulating common bean <100g1 soil

Traits NN NDW SDW NPP NSP 100 SW TBY GY HI

NNNDW 0.93***SDW 0.19 ns 0.28 ns

NPP 0.15 ns 0.39 ns 0.44 ns

NSP 0.11 ns 0.14 ns 0.59* 0.66*

100 SW 0.08 ns 0.21 ns 0.67* 0.78** 0.74**

TBY 0.68* 0.76** 0.69* 0.50* 0.56* 0.65*

GY 0.56* 0.70* 0.52* 0.46 ns 0.59* 0.58* 0.91***

HI 0.13 ns 0.04 ns 0.08 ns 0.05 ns 0.30 ns 0.08 ns 0.09 ns 0.00 ns

PTTN 0.13 ns 0.18 ns 0.23 ns 0.66* 0.72* 0.56* 0.36 ns 0.38 ns 0.52*

Ns non-signicant, NN-Nodule number per plant, NDW nodule dry weight per plant (g plant1), SDW shoot dry weight (g plant1), NPP number of pods per plant, NSP

number of seeds per pod, 100 SW 100 seed weight (g), TBY total biomass yield (kgha1), GY grain yield (kgha1), HI harvest index * Signicant at 0.05; ** highly signicant at 0.01; *** very highly signicant at 0.001

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Table 6 Correlation amongthe investigated traits ofcommon bean treated dierent rates ofN withand withoutinoculation insoil having rhizobial population nodulating common bean between100 and1000g1 soil

Traits NN NDW SDW NPP NSP SW TBY GY HI

NNNDW 0.96***SDW 0.60* 0.55*

NPP 0.40 ns 0.34 ns 0.77**

NSP 0.35 ns 0.26 ns 0.78** 0.66*

SW 0.26 ns 0.26 ns 0.51* 0.66* 0.34 ns

TBY 0.36 ns 0.29 ns 0.70* 0.88*** 0.57* 0.71**

GY 0.61* 0.64* 0.73** 0.81** 0.49 ns 0.69* 0.77**

HI 0.25 ns 0.29 ns 0.19 ns 0.26 ns 0.16 ns 0.46 ns 0.17 ns 0.50*

PTTN 0.42 ns 0.36 ns 0.77** 0.84** 0.72** 0.52* 0.91*** 0.69* 0.03 ns

ns non-signicant, NN-Nodule number per plant, NDW nodule dry weight per plant (g plant1), SDW shoot dry weight (g plant1), NPP number of pods per plant, NSP

number of seeds per pod, 100 SW 100 seed weight (g), TBY total biomass yield (kgha1), GY grain yield (kgha1), HI harvest index * Signicant at 0.05; ** highly signicant at 0.01; *** very highly signicant at 0.001

Table 7 Correlation amongthe investigated traits ofcommon bean treated dierent rates ofN withand withoutinoculation insoil having rhizobial population nodulating common bean>1000g1 soil

Traits NN NDW SDW NPP NSP SW TBY GY HI

NNNDW 0.72**SDW 0.45 ns 0.23 ns

NPP 0.45 ns 0.10 ns 0.87***

NSP 0.18 ns 0.29 ns 0.41 ns 0.27 ns

SW 0.00 ns 0.07 ns 0.68* 0.62* 0.05 ns

TBY 0.56* 0.27 ns 0.74** 0.79** 0.49 ns 0.50 ns

GY 0.75** 0.69* 0.75** 0.61* 0.55* 0.39 ns 0.77**

HI 0.33 ns 0.34 ns 0.08 ns 0.15 ns 0.28 ns 0.06 ns 0.49 ns 0.25 ns

PTTN 0.44 ns 0.02 ns 0.38 ns 0.53* 0.27 ns 0.03 ns 0.26 ns 0.09 ns 0.11 ns

Ns non-signicant, NN-Nodule number per plant, NDW nodule dry weight per plant (g plant1), SDW shoot dry weight (g plant1), NPP number of pods per plant, NSP

number of seeds per pod, 100 SW 100 seed weight (g), TBY total biomass yield (kgha1), GY grain yield (kgha1), HI harvest index * Signicant at 0.05; ** highly signicant at 0.01; *** very highly signicant at 0.001

In sites with soil having a low rhizobial population, GY of common bean correlated positively with SDW (r=0.5152; P0.05), NSP (r=0.5907; P0.05), 100 seed weight (r=0.5791; P0.05) and TBY (r=0.9121;

P0.001) (Table5). Similarly, strong and positive correlation was observed between GY with SDW (r=0.7271;

P0.01), NPP (r=0.8098; P0.01), 100 seed weight (r=0.6939; P0.05) and TBY (r=0.7713; P0.01)

in experimental soil with medium rhizobial population (Table 6). In soil showing a high rhizobial population, we noted a positive correlation between GY and SDW (r=0.7496; P0.01), NPP (r=0.6067; P0.050), NSP (r=0.5474; P0.05) and TBY (r=0.7712; P0.01)

(Table7). Similarly, Bayuelo-Jimnez etal. [64] had previously indicated a positive and signicant correlation among yield and yield components of common bean.

The correlation analysis indicated a positive relationship between PTTN and NPP (r=0.6644; P0.05), NSP (r=0.7195; P0.05) and 100 seed weight (r=0.5562;

P < 0.05) in experimental sites showing soil with low rhizobial population (Table 5). In those with medium rhizobial population, a positive and strong correlation between PTTN and SDW (r = 0.7665; P 0.01), NPP (r = 8386; P 0.01), NSP (r = 0.7154; P 0.01), 100 seed weight (r = 0.5206; P 0.05), TBY (r = 0.9114;

P0.001) and GY (r=0.6941; P0.05) (Table6). These results agree with the previous studies, which reported the signicant correlation between plant N accumulation with seed yield, seed weight and total biomass [45, 50]. Ruiz-Dez etal. [45] also demonstrated that plant N accumulation was the most suitable trait for the selection of highly eective and highly competitive Rhizobium

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isolates. In contrast, in soil with high rhizobial population, PTTN was only signicantly but inversely correlated with NPP (r=0.5296; P0.05), thus, indicating the negative impact of mineral N treatment on soil productivity, despite the plant N accumulation was noted (Table7). The present study also indicated a non-signi-cant relationship between PTTN and both NN and NDW in all soil types. Similar result was previously observed by da Silva etal. [24] who reported that the correlation between xed N and nodulation was decreased with increasing rates of N application. In contrast to this, Tsia et al. [25] found a positive and signicant relationship between both NN and NDW and N in shoots at 45days after emergency.

Conclusion

The results of this study indicate the signicant eect of N treatments on the nodulation, yield and yield traits of common bean in the major growing areas of eastern Ethiopia. Our experiments indicated the need of inoculation of Rhizobium beside that of a starting N dose in soil having <100 rhizobial population g1 of soil. An N requirement to reach the highest yield of common bean was not aected by the native rhizobial population. The eect of Rhizobium inoculation on nodulation of common bean appears to depend on the type of soil. Our data also indicated that the amount of mineral N required for maximum seed yield is dependent on the original soil rhizobial population. Moreover, the indigenous rhizobial population that nodulates common bean also aects the bean production. Further research on eectiveness of the combination between indigenous rhizobia and inoculated rhizobial population would be recommended.

Authors contributions

Both of us participated equally starting from the development of the research idea, writing proposal and competing research grant and development of this manuscript. But I participated more in the management and collectionof data from the eld experiment, which is why I am the rst author of this manuscript. Both authors read and approved the nal manuscript.

Author details

1 College of Agriculture and Environmental Sciences, School of Natural Resources Management and Environmental Sciences, Haramaya University, Dire Dawa, Ethiopia. 2 Ethiopian Institute of Agricultural Research, Holleta Agricultural Research Center, Holleta, Ethiopia.

Acknowledgements

This work was carried out with the support of Ethiopia Institute of Agricultural Research under National Biofertilizer Development Project hosted at Holleta Agricultural Research Center, Soil microbiology research group. The author also thanks Berhanu Mengistu, Dejene Ayenew and Girmaye Mekonnen for managing the eld experiments.

Competing interests

The authors declare that they have no competing interests.

Received: 19 October 2015 Accepted: 21 October 2015

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