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	�:	Evaluation of soybean nodulating rhizobia strains under field condition at    soybean growing areas of South Western Ethiopia.
Corresponding author: Reshid Abafita Abawari, Jimma agricultural research center, Jimma,  Ethiopia,
E-mail:  HYPERLINK "mailto:reshida5@gmail.com" reshida5@gmail.com P.O. Box 192, Jimma, Ethiopia
Abstract:
 Because of the rising costs of chemical fertilizers and the growing environmental concerns, there is a need in the use of bio-inoculants in crop production and soil fertility enhancement. A field study was therefore conducted at Jimma zone kersa wereda during 2014 main cropping season to evaluate soybean nodulating rhizobia strains under field condition in order to select the best performing rhizobia strain for nodulation, yield and yield attributes of soybean. Eight treatments were arranged in RCBD with three replications. Nodule number, plant height, number of pods per plant, dry biomass and grain yield was responded significantly to the interaction effects of TAL-379 inoculation. The maximum mean yield of 22quintal per hectare was obtained from TAL-379 inoculation. Similarly the highest mean nodule number of 42.67 and 35.47 per plant was counted after inoculation with TAL-379 and SB-12 strains respectively. Moreover the highest mean plant heights of 65.67cm followed by 62.67cm were measured by inoculation of SB-12 and TAL-379 strains respectively. These results demonstrated that the dominance of TAL-379 and SB-12 rhizobium strains and could explain the effectiveness of the strains in South west soil of Ethiopia. Inoculation of TAL-379 and SB-12 strains with Clark 63-K soybean variety was being considered effective symbionts for Clark 63-K soybean production in the area.  
Key words: Dominant Rhizobia, Growth, Nodulation, soybean variety, Jimma zone.

Introduction
There is no doubt on the need to address the problem of low soil fertility in Ethiopia in order to improve agricultural productivity and uphold the rapid growing human population. However, the cost of chemical fertilizers and their associated risks on the environmental safety calls for the search of alternative means of plant nutrient management practices (ESS, 1993). Accordingly, production potential of grain legumes and other cereal crops are still low and as a result producers are now looking for other alternatives to these fertilizers. Urea, which is the most commonly used nitrogenous fertilizer, has now become a costly input for most of the farmers. As such, rhizobia inoculants may be used as a cheaper substitute for urea in the production of food legume crops (Karim, et al., 2001).  Legumes are assumed to be self-sufficient for all of their nitrogen requirements, when they are inoculated with effective nitrogen fixing strains of rhizobia. Bradyrhizobium, Mesorhizobium and Sinorhizobium nodulate soybean and fix nitrogen symbiotically. The beneficial effect of rhizobial inoculants in increasing yield of leguminous crops results from the activity of its root nodule bacteria, which fix atmospheric nitrogen making it available for the plants and thus not only increase the production of inoculated crops, but also leave a fair amount of nitrogen in the soil, which benefit the subsequent crops. Ellafi et al., (2011) reported that legumes have been an essential component of crop production since ancient times because of their role in improving soil fertility via biological Nitrogen fixation. Legumes play an important role in improving food and nutrition security. Legume seeds contain double or triple the amount of proteins as cereal grains do, provide calories and essential micronutrients and are low in fat and high in fiber (FAO, 2016); and hence they are quickly becoming an important part of farming systems in Africa. They are the only sources of this nutrition for the vast majority of poor producers and consumers in developing countries (Nedumaran et al., 2015). According to�Central Statistics Authority {CSA} (2000), in Ethiopia Legumes rank second after cereals and occupy 17.7% of the total of the total cultivated areas and contribute about 12% of the total production. The major food legumes produced in Ethiopia are faba bean, field peas, soybean, lentil, chickpea, haricot bean, fenugreek, cowpea, pigeon pea, grass pea and lupine. Among legumes, soybean (Glycine max), is one of the most important and oldest cultivated crops (Martin and Leonard 1967). It is a summer annual herb that has never been found in the wild (Hirth, 1908). However, production per unit of land is very low because of adoption has been limited due to weakly developed marketing channels, poor quality assurance, running the risk of losing confidence of farmers in the product and inadequate capacity within the extension services (Bala et al., 2011).  
Feasibility of biological nitrogen fixation in Ethiopia has been well reviewed and documented (Hailemariam and Tsige, 2006). This successful symbiotic association requires the survival of rhizobia in sufficient numbers as free living bacteria in the soil ecosystem (Crozat et al., 1982).  
   Bio-inoculants as an alternative to commercial fertilizer N for pulses are gaining priority due to its economical and ecological benefits (Jensen et al., 2012). This demand has been created apparently since 2012 in areas of South Western Ethiopia, where rhizobia inoculants product has been disseminated. Nevertheless, isolation and selection of the most effective rhizobial strains for soybean growing areas of South Western Ethiopia, creation of large scale awareness, use and importance of bio-inoculants technologies are yet to be done throughout the region. Therefore elucidation of this phenomenon and the development of competent and effective cultures for legume inoculants should be the major contributions of researchers to soil science.  
   MATERIALS AND METHODS
The experiment was established in the 2014 cropping season under rain-fed condition at the South western part of Ethiopia. The field experiment was conducted on on-station, located in the south western Ethiopia, Jimma zone (Karsa wereda). The soil had not been inoculated before with rhizobia strain nodulating soybean. 
Seed of Clark 63-K, a well adapted soybean cultivar in the area with 95% viability, was obtained from Jimma Agricultural Research Center and used as a test crop. The variety was selected based on their yield, their maturity time, and recently improved released varieties of the experimental region.  The experiment was carried out in a randomized complete block design (RCBD) with eight treatments and three replications.    
  Strain of Rhizobium inoculants;SB-6B1, SB-6A2, SB-Murd, SB-12, were obtained from Holeta Agricultural research center soil microbiology laboratory and TAL-379 was obtained from Menagesh Biotech private limited company (Ethiopia). DAP and Triple super phosphate (TSP) was obtained from Jimma Agricultural Research Center and used as positive control. 
The size of each plot was 4m� 3.6m=14.4m2, with total harvestable plot area (Net) 4mx0.6mx4 = 9.6m2. The space between plots was 1m and the space between the blocks was 1.5m. Each plot contained 6 planting rows and the space between rows was 0.60m. At planting, two soybean seeds were seeded at 5cm distance within a row and at 5cm depth. The middle four rows were used for data collection. Seeds were hand planted in rows from June 18-20/2014. DAP was used as N source for positive control while P was applied as triple super phosphate (TSP).
 The composite soil sample was analyzed for selected physical and chemical properties. Texture was determined by the Hydrometer principle whereas soil pH was measured from the suspension of 1: 2.5 soils: H2O by pH meter. Soil organic carbon was determined by the Wakley and Black (1934) and FAO (2008) method and available phosphorus in the soil was determined based on (Olsen et al. 1954) (Table 1).    
Soybean seeds were   inoculated by covering them with paste of inoculum which was made from a rate of 10 g of peat-based powder inoculants per 100 g (Somasegaran and Hoben, 1985; Deaker et al., 2004) of seed just before planting.  
Table1.Physico-chemical properties of the soil in the study site before planting of soybean during 2014 cropping season.
Measured indexes	Physico-Chemical properties of the soil Soil  Type                                 Clay%Clay                                      40.5% silt                                        11.5%sand                                        41.5PH                                4.10 N (1gm %N)   0.19 P(ppm)1.75 K  (Meq K/100gm)                                      2.18 Organic matter (%)                  7.50Organic Carbon (%)               4.40 Exchangeable acidity(Meq K/100gm) 0.49
Data Collection and Analysis
Five plants for destructive data were randomly sampled from the two border rows of each plot at mid flowering during the maximum growth stage. Then the soil particles were removed gradually by washing the roots with water. Total number of nodules per plant, shoot fresh and dry weight and plant height were recorded. Number of pods per plants, number of seeds per pods, grain yield and moisture content were determined during harvesting time. Grain yield was corrected for 10% moisture content using Draminski moisture meter and converted in to kilogram per hectare. Analysis of variance (ANOVA) was conducted using the General Linear Model procedure of Statistical Analysis System (SAS, 2002) software version 9.0 and Least Significant Difference method at 0.05 probability level was used for mean separation (Gomez and Gomez, 1984).
RESULTS 
Soil Physical and Chemical Properties: The soil texture of the study site was clay. The soil pH of study site was 4.10 (pH in water at soil: liquid ratio of 1:2.5) (Table 1).  The organic carbon, organic matter and the extractable phosphorus concentration recorded from study site were 4.40%, 7.50% and 1.75ppm respectively, which are in the very low range (London, 1991, FAO., 1990). The potassium concentration of the study site was 2.18% which is in the high range (Table 1) (London, 1991).The total N concentration of the study site was 0.19% (Table 1), which is in the low range (London, 1991, FAO., 1990).  
Effects of Inoculation on Number of Nodules and Pods per Plant and Number of Seeds per Pods: The mean number of nodule per plant from each experimental plot is indicated in Table 2, showing that they varied among treatments. Significantly higher mean numbers of nodules per plant was observed from soybean planted with TAL-379 (42.67) followed by SB-12 (35.42).inoculants as compared to those planted with the rest rhizobia strains. The lowest values were observed from treatments SB-6B1, SB-6A2, SB-14 and SB-Murd. This indicates that the available local strains were more competitive and effective as compared to inoculated rhizobia strains of SB-6B1, SB-6A2, SB-14 and SB-Murd. Other inoculums such as SB-14, SB-6B1, SB-6A2, and SB-murd have no significant effects on number of nodules per plants during on-station trials (Table 2). The results of analysis of variance indicated that application of TAL-379 had significant effect (P<0.05) on mean pod number per plants (22.67 per plant). On the other hand, application of SB-14, SB-6B1, SB-12 and SB-Murd resulted in better mean pod number per plants than the mean pod number obtained by application of SB-6A2 and the control (Table 2).  
The results of analysis of variance indicated that the interaction of all inoculated strains did not significantly affect the seed yield per pods. However, there were slight differences among seed inoculation of rhizobia to seed yield per pods and interactions of P as well as N fertilizer.
   Soybean planted with rhizobia inoculation (SB-12 and TAL-379) produced higher mean numbers of nodules per plant (Table 2). Inoculation of soybean with TAL-379 strains had significant effects over mean number of pods per plants. Moreover, except SB-6A2strain, higher number of pods per plant was observed from soybean planted with rhizobia inoculation such as SB-14, SB-6B1, SB-12 and SB-Murd.
Effects of Inoculation on Plant Height and Dry Biomass: The results of analysis of variance indicate that the interaction of SB-12 was significant (p<0.05) on plant height (65.67cm).The highest mean plant height was recorded from SB-12 rhizobia strains. Similarly a comparable mean plant height was obtained by inoculating SB-Murd and TAL-379 strains. Moreover, the use of 100kg ha-1 DAP alone significantly improved the mean plant height.  
A significant mean dry biomass was obtained from soybean planted with inoculation of TAL-379, SB-Murd and SB-12 compared with inoculation of other strains.  Mean dry biomass of 7.87, 7.77 and 7.23 tons per hectare followed by 6.3 tons per hectare were obtained from TAL-379, SB-Murd, SB-12 and SB-14 respectively, indicating variations among strains.   The lowest dry biomass was obtained from inoculants SB-6B1 and SB-6A2 (Table 2).   
Effects of inoculation on grain yield and field weight: The results of analysis of variance showed that the response of grain yield and field weight to the interaction effects of TAL-379 was significant (P< 0.05). Soybean seed inoculation with TAL-379 produced higher mean grain yield (22 quin ha-1) and field weight (58.8quin ha-1) followed by SB-Murd, SB-12 and SB-14 than that of SB-6A2 and the uninoculated control (Table 2). The highest grain yield was recorded when the TAL-379 (rhizobia strain) interacted with Clark 63-K soybean variety. 
 Table  (2). Soybean yield and other yield components affected by application of inoculums at kersa on-farm during 2014/2015 crop season.     
TreatmentsNo. of nodule/plantStand countplant ht( cm)No. of pods/plantseeds/podField  Wt.qui/hagrain yield qui/haMoisture (%)Biomass Yield  Tone/haSB-6A2 0.80b490.67c      50c15b2.33a      38.43b15.17c16.3a5.2bSB-14 1.13b571.67a      58abc19.33ab      2.67a      47.92ab16.73abc16.07ab6.3ab      SB-Murd 2.93b489.67c      61.67ab      20.33ab      3a58.33a21.45ab      15.73ab7.77a      SB-6B1 4.47b491.67c      54.33bc      17.33ab      2.33a      38.43b14.08c15.77ab5.13b      SB-12 35.47a514.33bc      65.67a      21ab2.67a      56.25a17.65abc15.7ab7.23a     TAL-379 42.67a543ab62.67ab      22.67a      2.33a      58.80a22.00a15.23ab7.87a      100kg/ha DAP2.13b512.33bc      63.67ab      14.67b     2.67a      46.30ab14.90c15.03b6ab100kg/ha TSP0.07b551.33ab      54.33bc      15b2.67a      47.22ab16.31bc15.9ab6.17ab      LSD(0.05)19.842.6510.536.980.9715.145.451.21.98CV101.134.68     10.2221.93     21.54      17.66     17.99      4.4517.5      Means followed by the same letter in a column are not significantly different at P = 0.05; TSP=Triple supper phosphate, No = Number, ht = height, wt = weight, qui = quintal, ha = hectare, CV = Coefficient of variation, LSD = Least significant difference, Ton=Tone.
DISCUSSION
 The soil reaction of the study site was strongly acidic based on pH in H2O (London, 1991). Most of the farmers do not realize that ammonium fertilizers tend to lower the soil pH resulting in acidity due to the microbial oxidation of ammonium to nitric acid (Nyalemegbe, et al. 2009). Therefore, use of lime, planting of acid-tolerant crops and integrated use of lime with organic fertilizer are recommended to amend the soils for profitable agricultural crop production. The relatively lower soil organic carbon, organic matter, extractable phosphorus concentration and Nitrogen of study site could be attributed to the continuous cropping and cultivation, intensive tillage practice and heavy rainfall in the area. This revealed that the requirements of the   use of supplementary fertilizers and organic amendments to optimize crop yields.
Therefore, biological N2-fixation needs to be an optional input for sustainable crop production and to combat soil acidity stress. Inoculation with effective rhizobia inoculants could stimulated legume plant growth through effects on nodulation and biological N2-fixation. This was justified with the finding of Dubey (1998) who obtained highest grain yield of soybean when the plant was inoculated with B. japonicum. Moreover, optimum growth of leguminous plants is usually dependent on symbiotic relationships with N2-fixing bacteria (Guo et al.2010; Xavier and Germida, 2003). Continuous cultivation may be necessary to help the build-up of rhizobia in soil inoculating such strains, resulting in increases in nodulation (Raposeiras, et al. 2006). The significant improvement in mean pod number per plants was due to the use of effective strains. This revealed that the inoculated rhizobia strains were more effective as compared to the pre-existing strains available in the soil.  Lower response to added inoculants may be due to failing to compete with the presence of indigenous rhizobia and the soil PH stress (Kutcher,et al.2002). Mungai and Karubiu (2012) stated that presence of high numbers of indigenous rhizobia may have limited nodule formation by introduced strains. The process of nodulation may be promoted by relatively low levels of available nitrate or ammonia, but higher concentrations of N almost always depress nodulation.   Therefore, inoculation of soybean seed with effective and efficient rhizobia strains under optimum environmental condition (Temperature, moisture and soil PH) is very crucial for economically feasible and sustainable production of soybean. 
The increase in mean plant height, mean dry biomass, grain yield and field weight could be attributed to increase in yield components of the crop through competent rhizobia inoculated. This was justified with the finding of Egamberdiyeva et al. (2004) who obtained highest grain yield of soybean when the plant was inoculated with B. japonicum in Uzbekistan.  The final grain yield of the crop is a function of cumulative contribution of its various growth and yield parameters which were influenced by competent rhizobia inoculated and various agronomic practices. Different strains presently studied varied in their effectiveness with respect to soil fertility status and their competency over indigenous soil rhizobia. Keneni et al. (2010) found the native rhizobia strains of the Wollo region to be more competitive than the exotic rhizobia strains. Therefore, planting of soybean without inoculation is recommended for soybean production where farm history showed that soybean had been grown previously. Furthermore, appropriate strain selection is recommended to identify the effectiveness and competitiveness of exotic rhizobium as compared to locally available rhizobia strains in a soil.
 CONCLUSION
The results of this study revealed that seed inoculation of TAL-379 and SB-12 could be the best strains for studied site and improve nodulation potential of soybean. The results obtained in this work might have potential applications for increasing the productivity of soybean and enriching the soil with Nitrogen. In fact, further knowledge on criteria for selection of effective rhizobia will allow us to better understand to select efficient isolates that can be used in inoculation projects to promoting the plants growth.
ACKNOWLEDGEMENTS
This work was supported by the Laboratory of jimma agricultural research center soil and plant tissue testing for their assistance and support. We would like to thank the staff members of the Jimma Agricultural Research Centre (JARC), for their material support and facilitating the field studies.  
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