Symbiotic Effectiveness of Elite Rhizobia Strains Nodulating Desi Type Chickpea (Cicer arietinum L.) Varieties
Assefa Funga1, 2, *, Chris O. Ojiewo3, Losenge Turoop2, Githiri Stephen Mwangi2
1Ethiopian Institute of Agricultural Research, Debre Zeit Agricultural Research Center, Bishoftu, Ethiopia
2Department of Horticulture, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya
3International Crop Research Institute for Semi-Arid Tropics, Addis Ababa, Ethiopia
To cite this article:
Assefa Funga, Chris O. Ojiewo, Losenge Turoop, Githiri Stephen Mwangi. Symbiotic Effectiveness of Elite Rhizobia Strains Nodulating Desi Type Chickpea (Cicer arietinum L.) Varieties. Journal of Plant Sciences. Vol. 4, No. 5, 2016, pp. 88-94. doi: 10.11648/j.jps.20160404.15
Received: May 29, 2016; Accepted: July 12, 2016; Published: August 12, 2016
Abstract: Chickpea (Cicer arietinum L.), is a multi-functional crop with important role in the diet as affordable protein source and in sustaining soil fertility through nitrogen fixation. However, its productivity in Ethiopia of 1.9 t ha-1 is lower than its potential of 5 t ha-1 under well managed conditions, partly due to soil fertility limitations. Field experiments were conducted to evaluate effectiveness of elite rhizobia strains on productivity of chickpea. Four rhizobial inoculant treatments and one control with three chickpea varieties were used. Inoculated plants had significantly (p<0.05) better performance with most of the symbiotic traits, grain yield and yield related traits than non-inoculated treatments. Shoot nitrogen yield was increased in the range of 13.0 – 31.34% by inoculation with strain ICRE-025 over the two test sites. The highest level of N fixation was achieved in genotype ICC-4918 by inoculation with EAL-029 and ICRE-025. Investigations at both test sites demonstrated that inoculation of chickpea varieties with native rhizobial strains were effective and useful for optimized chickpea production.
Keywords: Desi Chickpea, Symbiotic Effectiveness, Nitrogen Fixation, Rhizobial Inoculant
Chickpea (Cicer arietinum L.) is a major grain legume cultivated for its edible seeds. Across the world chickpeas are mainly cultivated in the cool, dry season of the semi-arid tropics on residual moisture . In Ethiopia, chickpea is mainly grown in the central, northern and eastern highland areas of the country at an altitude range of 1400-2300 m.a.s.l., where annual rainfall of 500 - 2000 mm is received on vertisols with good water holding capacity [1, 2]. Ethiopian farmers essentially plant chickpea on residual moisture after the end of the main rainy season, usually in late August – October .
Chickpea, a multi-functional crop, has an important role in the diet of the Ethiopian small scale farmers’ households and also serves as protein source for the rural poor who cannot afford to buy animal products . The crop also serves as a source of cash income and plays a major role in Ethiopia’s foreign exchange earnings through export to Asia and Europe . Due to its capacity of biological nitrogen fixation; chickpea can improve the soil fertility status [3, 5]. Recent research shows that chickpea can fix more nitrogen than other pulse crops, thus enhancing soil fertility for subsequent crops . The Rhizobium bacteria that are compatible for nodule formation on the roots of chickpea are different from those that nodulate peas and lentils. Thus inoculating chickpea seed with the correct inoculant is critical.
In spite of all these merits and benefits, the productivity of this crop remains low in Ethiopia. While yields of up to 5.5 tons ha-1have been obtainedon experimental stations in Ethiopia , the latest average national productivity reported is as low as 1.9 tons ha-1 . This yield gap between average and potential yield of chickpea could be due to many factors, of which environmental constraint associated with the absence of specific and effective rhizobia in the soil is one of the most important  that affect the productivity of the crop.
Different reports indicate that chickpea fixes atmospheric nitrogen in the range of 60 - 140kg N ha−1 in one cropping season [10, 11, 12]. There is increasing evidence that suggests that more nitrogen can be fixed by existing legume grain crops if they are inoculated more often or with more effective strains of Rhizobia . Additionally Ayaz et al  reported that inoculation of seed with its own specific and suitable Rhizobium strain before planting is crucial to fully benefit from grain legume crop in terms of maximum yield and soil improvement.
Ethiopia has a great potential of benefiting from enhanced BNF that may contribute to increased yield and sustained soil fertility. This is because larger area is devoted to grow chickpea as compared to other African counties ; there are many rhizobia strains that have been identified and proven to be effective and competitive ; there are also a number of high yielding released varieties . However, use of high yielding varieties alone may not result in the required level of productivity without involvement of compatible and effective rhizobia strains [9, 17].
A given legume cultivar nodulated by different strains of the same species of rhizobium would fix different amounts of nitrogen. And it is also true that a given strain of Rhizobium will nodulate and fix different amount of N in symbiosis with a range of cultivars of the same plant species . Keneni et al  has also suggested that there may be the need to conduct specific tests for specific breeding materials, strain and environments to improve the selection process to gain host genotype compatible to specific rhizobia strain. However, our knowledge on effective rhizobia strains for efficient symbiosis with chickpea varieties is scanty. Besides, the efforts to test and recommend rhizobia inoculums on-farm have been limited. Chickpea farmers therefore traditionally produce the crop without the use of biofertilizers. This study was conducted to evaluate effectiveness of elite rhizobia strains on productivity of improved chickpea (Cicer arietinum L.) varieties at Debre Zeit and Wolayta Sodo, Ethiopia.
2. Material and Methods
2.1. The Test Environment
The experiment was conducted under field conditions at Debre Zeit (representing the central highlands) and Wolayta Sodo (representing southern region) of Ethiopia during the main cropping season of 2015/2016 (September–January). Soil samples from both locations were collected from the rhizosphere at the depth of 0 to 30 cm for physico-chemical characterization (table 1).
|Parameters||Debre Zeit||Wolayta Sodo|
|Altitude (m, a. s)||1900||1880|
|pH (1:1.25 H2O)||6.61 (Slightly acidic)||6.00 (Slightly acidic)|
|Organic Carbon (%)a||1.2 (Low)||1.67 (medium)|
|Organic Matter (%)||2.1 (Low)||2.87 (Low)|
|Total Nitrogen (%)b||0.16 (medium)||0.19 (medium)|
|Available phosphorus (ppm)c||73.25 (excess)||9.17 (Low)|
|Exch K (cmol)/kgd||0.40 (High)||0.23 (High)|
|CEC (cmol)/kge||39.89 (High)||19.70 (Medium)|
|Moisture content (%)||4.54||1.49|
Method: a = Walklay and Black; b = Kjeldahl; c = Olsen; d &e = Ammonium acetate; f = Hydrometer; ppm = parts per million
2.2. Treatments and Layout
The experiment was conducted using three chickpea verities (Natoli, Teketay and ICC-4918) with four levels of rhizobia strains (EAL-029, ICRE-025, ICRE-03 and ICRE-05) and a control (non-inoculated) were used in split plot design with three replications. Chickpea varieties as main plot and the four rhizobium inoculants and a control as sub plots were assigned to experimental units randomly. Each of the main plots was divided into five sub plots having plot size of 4m by 1.2m (4.8m2). The spacing of 30 cm and 10 cm was used between and intra row respectively. Accordingly, each sub plots consisted of four rows out of which the outer two rows were used as border rows and in each rows 40 – 45 seeds were planted. Phosphorus fertilizer in the form of TSP was applied to all plots at the rate of 100 kg ha-1. All other crop management and protection practices were applied uniformly to all experimental units as required.
2.3. Inoculants Preparation and Method Used
Three elite Rhizobia strains (ICRE-025, ICRE-03 and ICRE-05) proven to be effective during greenhouse and laboratory studies were obtained from the soil microbiology laboratory of the School of Plant and Horticultural Sciences, Hawassa University, Ethiopia and the commercial biofertilizer EAL-029 was obtained from National Soil Laboratory of Ethiopia. All the necessary aseptic precautions were taken when preparing inoculums and inoculation. Accordingly, using peat based inoculation method at the recommended rate of 10g per kilogram of seed, 200g of chickpea seed was soaked in 30 ml of 5% sugar solution. The contents were stirred well. Sugar solution improves the adhesion of inoculant to the seed. Then 2g of inoculant was added on the wetted seed and mixed in a plastic bag until all the seeds were uniformly coated. The whole inoculation procedure was completed in shade as sunlight damages the bacteria. Seed was sown immediately after inoculation.
2.4. Soil Characteristics of the Study Sites
The physico-chemical properties of the soils from the two test locations, Debre Zeit and Wolayta Sodo, showed medium levels of nitrogen contents (0.16 and 0.19%) but high levels of K. The levels of CEC (39.89 cmol kg-1) was high at Debre Zeit and medium (19.70 cmol kg-1) at Wolayta Sodo with pH values slightly acidic ranging from 6.0 to 6.61 which is within the optimum pH range for crop production (6.0 - 8.2) . It was also found to be favorable for chickpea infective strains of Rhizobia . The level of soil phosphorus was excessively high at Debre Zeit (73.25 ppm) and low at Wolayta Sodo (9.17 ppm) (Table 1). Generally soils at the two experimental sites showed minor differences in their content of major nutrients and characteristics, with Debre Zeit soil somewhat more fertile than Wolayta Soda soil.
The amount of rainfall received by the two test sites was nearly the same within the growing season of chickpea. Debre Zeit received 228 mm while Wolayta Sodo received 187 mm rainfall from August to December 2015 (figure 1). However, it was much lower than the minimum requirement (500 mm) reported by Bejiga and van der Maesen . During the study period the weather variables recorded deviated much from the long-term trends at both locations, causing irregular distribution and low amount of rainfall. The growing season was generally characterized by erratic form of rainfall and hence growth of chickpea and activity of micro organisms in the soil to some extent might be adversely affected.
NB: DZ MAXT= Debre Zeit max. temperature, DZ MINT= Debre Zeit min. temperature,
WS MAXT= Wolayta Sodo max. temperature, WS MINT= Wolayta Sodo min. temperature
2.5. Data Collection
Nodulation was assessed at the mid-flowering stage of chickpea. Five representative plants were randomly taken from the border row on each side of the plot for nodule number and nodule dry weight determination. Crop phenological data including days to 50% flowering (DF) and days to 90% maturity (DTM) were also taken at respective growth stages. Five plants from each plot were sampled at the time of harvest and separated into grain and straw for plant tissue analysis for nitrogen content. The straw part was oven dried at 70°C for 48 hours, milled with mechanical miller and passed through a 1mm sieve. Subsequently, the plant straw was analyzed for total N. The straw total N uptake was calculated as a product of their respective straw yield obtained from five plants and N content obtained from the lab analysis i.e., N uptake (shoot dry weight x N concentration) plant-5. Nitrogen fixed was determined using the procedure formulated by Yaman and Cinsoy  as follows: N fixed (N uptake in an inoculated variety – N uptake in uninoculated variety) were calculated. Thus, the following data were recorded as: shoot nitrogen content (SHNC), shoot nitrogen uptake in gm plant-5 (SHNY). Percent change (increase or decrease) in data of nodulation traits, grain yield and nitrogen uptake for inoculated treatments was obtained by the formula given below.
Where, = Mean of inoculated variety, and = Mean of uninoculated variety
2.6. Data Analysis
Statistical analysis of data collected from the field and laboratory was analyzed using mixed linear model procedure of SAS 9.3 after checking the compliance of the data with the assumptions of the statistical test. Mean separation test was done using LSD at p-value 0.05.
3.1. Effect of Inoculation on Nodulation Performance of Chickpea
Inoculation of chickpea varieties by Rhizobial strains affected significantly (p < 0.05) nodule count plant-1 (NCPP), nodule dry weight (NDW) at Wolayta Sodo. Inoculation did not affect the nodulation status and nodule dry weight of chickpea at Debre Zeit. However, inoculated varieties produced higher number of nodules over control treatments. In the experiment conducted at Wolayta Sodo, varieties inoculated with strains ICRE-03 produced the highest nodule dry weight (0.29 gm plant-1), followed by those treated with strains EAL-029 (0.28gm plant-1) and ICER-05 (0.28 gm plant-1) (table 3).
3.2. Effect of Inoculation on Symbiotic Nitrogen Fixation of Chickpea
Shoot nitrogen content (SHNC) at Wolayta Sodo and shoot dry weight (SHDW), and shoot nitrogen yield (SHNY) were also affected significantly (p<=0.05) at Debre Zeit. Shoot nitrogen content (SHNC) of the chickpea varieties was not affected by inoculation at Debre Zeit. However, inoculated varieties had higher nitrogen content over control treatment. Similarly, in an experiment conducted at Wolayta Sodo the highest shoot nitrogen content was recorded in varieties inoculated with strain ICRE-025 (0.62%) followed by ICRE-03 (0.61%).
Rhizobium inoculation affected shoot dry weigh of chickpea varieties at Debre Zeit and increased by 5.7 -16.0% over control. The highest increment was achieved by inoculation with strain ICRE-03. At Wolayta Sodo inoculation did not affect shoot dry weight significantly but varieties inoculated with Rhizobia strains had 9.4 - 21.6% higher shoot dry weight than the control (Table 3).
Shoot nitrogen yield (SHNY) was also affected by rhizobial inoculation at both test sites. In the experiment conducted at Debre Zeit the highest shoot nitrogen yield (23.7 gm plant-5) was recorded from varieties inoculated with Rhizobia ICRE-03, followed by strain EAL-029 (23.24 gm plant-5). Similarly, at Wolayta Sodo the highest shoot nitrogen yield (32.1 gm plant-5) was recorded from varieties inoculated with rhizobia ICRE-025, followed by strain ICRE-03 (30.8 gm plant-5) (Table 2 and 3)
|Variety||Rhizobia strain||NCPP||NDW||DTM||SHDW||SHNC||SHNY||N fixed||RE (%)|
|------------------------------------------Rhizobia strains ------------------------------------|
|P-value (< 0.05)|
|R x V||ns||ns||ns||ns||ns||Ns||ns||Ns|
NCPP = nodule count plant-1, NDW =nodule dry weight plant-1, DTM= days to 90% maturity, SHDW = shoot dry weight gm plant-1, SHNC = shoot nitrogen content (%), SHNY = shoot nitrogen yield gm plant-5, N fixed = nitrogen fixed gm plant-5 and RE (%) = Relative effectiveness (%); ** = Significant (P <= 0.01), * = significant (P <= 0.05) and ns=non-significant (P> 0.05), V= Variety, R= Rhizobia strain and V*R= the interaction of variety and Rhizobia strain, Ino-mean = mean of inoculated treatments. Means with the same letter are not significantly different
|Variety||Rhizobia strain||NCPP||NDW||DTM||SHDW||SHNC||SHNY||N fixed||RE (%)|
|----------------------------------------- variety ----------------------------------------|
|---------------------------------------- Rhizobia strains --------------------------------|
|P-value (< 0.05)|
|R x V||ns||ns||ns||ns||*||ns||ns||ns|
NCPP = nodule count plant-1, NDW =nodule dry weight plant-1, DTM= days to 90% maturity, SHDW = shoot dry weight gm plant-1, SHNC = shoot nitrogen content (%), SHNY = shoot nitrogen yield gm plant-5, N fixed = nitrogen fixed gm plant-5 and RE (%) = Relative effectiveness (%) ** = Significant (P <= 0.01), * = significant (P <= 0.05) and ns=non-significant (P> 0.05), V= Variety, R= Rhizobia strain and V*R= the interaction of variety and Rhizobia strain, -mean = mean of inoculated treatments, Means with the same letter are not significantly different.
4.1. Effect of Inoculation on Nodulation Performance of Chickpea
In the present study all chickpea varieties showed nodulation in uninoculated treatment, confirming the presence of pre-existing native Rhizobia in the soil. Thus significant variation as a result of inoculation was observed among the genotypes for nodulation traits at Wolayta Sodo (Table 3). Though significant variations were not observed for nodule count (NCPP) and nodule dry weight (NDW) at Debre Zeit, inoculated varieties produced higher NCPP, NDW (Table 2). There was a strong genotypic/varietal effect on nodulation and nitrogen fixation in this study. Genotype ICC-4918 was included to the experiment as non nodulating check but was found to be nodulating. Regarding non nodulating genotypes of chickpea, there are recently published reports of findings which are contradicting. Gul et al  reported that the four genotypes (ICC 4993, ICC 19183, ICC 4918 and ICC19181) purchased from ICRISAT did not produce any nodule in control as well as inoculated treatment while other treatments produced nodule even without inoculation. On another finding Gul et al  also point out that ICC-4918 was late maturing and low yielding genotype being used as non nodulating check. In contrast to this Keneni et al  reported that a non nodulating genotype ICC-19180 was found to be nodulating with best performance for grain nitrogen yield and assimilation efficiency of fixed nitrogen and suggested that whether a change in environment alone can induce nodulation of a genotype that is naturally non-nodulating in another environment needs to be investigated in the future.
In the current study the nodules were observed and collected not only from inoculated seed of the genotype ICC-4918 but also was from the uninoculated plots where ICC-4918 was planted at both test sites. The findings from this study supports the suggestion given by Keneni et al  but postulates that the reason for ICC-4918 failing to nodulate in earlier studies in India was basically due to lack of compatible Rhizobia strains. It is plausible to conclude here that the efficiency of nodulation and nitrogen fixation depends on the compatibility between the chickpea genotype and the Rhizobia strain and the environmental conditions mostly soil factors.
4.2. Effect of Inoculation on Symbiotic Nitrogen Fixation of Chickpea
Rhizobial inoculation affected significantly (p<= 0.05) shoot dry weight and shoot nitrogen yield both at the two test sites. However, the response to inoculation was higher at Wolayta Sodo for most of measurements of symbiotic performances. For instance mean performance of shoot nitrogen content (0.59 %) was higher than that of SHNC (0.42 %) at Debre Zeit. SHNY (28.99 gm plant-1), amount of nitrogen fixed (5.11 gm plant-5) and relative effectiveness in terms of percentage of fixed nitrogen increased over uninoculated treatment (23.75%) were also higher than that of Debre Zeit (18.35%) (Table 2 and 3). The reason could be due to less fertile soil (table 1) and low presence of indigenous population of Rhizobia at Wolayta Sodo . Imran et al  also reported that inoculation response was more significant in soils having lower indigenous rhizobial population and fertility. On the other hand Debre Zeit was less responsive to inoculation for symbiotic characters of chickpea. Soil fertility status (table 1) has great impact on the response of chickpea variety to inoculation. In fact, Debre Zeit is the center of chickpea breeding and production for many years and hence there might be high rate of native rhizobia population in the soil capable of hindering the development of the new test Rhizobia strains used in the present study. There have been several reports by many authors such as [25, 26, 27, 28] that the presence of native Rhizobia is a major problem for inoculant performance under field conditions.
Depending on variety and Rhizobia strain used, the percentage of fixed nitrogen increased over control (uninoculated treatment) of the two test sites were from 13.01- 31.34% (Table 2 and 3) in chickpea shoot dry weight. The potential of fixation by the genotypes may be generally limited because of a shortage of soil moisture as chickpea crop normally grown on residual moisture . Moreover, the climate of the growing season deviated from the long term trend due to El Niño effect that caused short and erratic rainfall resulting in longer dry periods that might have affected the development of crop and soil micro organisms. Beck & Rupela  reported that fixation in winter-sown chickpea reached over 80–81%, whereas spring-sown chickpea, where moisture was a limiting factor, fixed only 8–27%. Additionally, Bergersen  reported that the nature of soil Rhizobial populations may affect the nitrogen fixation potential of legumes.
To have successful inoculation with an effective isolate that would result in an enhanced nitrogen fixation, one or both of the following conditions should be in place. First, the number of available invasive Rhizobia may be insufficient to nodulate the host adequately. Second, the average effectiveness of the population in the soil has to be inadequate to support the host's fixed nitrogen requirements . Difference in method of determination of the amount of nitrogen may also underestimate the amount of nitrogen fixed . Rhizobial inoculation also affected other associated characters of symbiotic nitrogen fixation. Chickpea varieties inoculated with strain EAL-029 produced higher nodule number, nodule dry weight and shoot nitrogen content followed by strain ICRE-03 at par for the two test sites. High fixed nitrogen in the shoot part of chickpea and high percentage increase of fixed nitrogen over the control treatment was also obtained from varieties inoculated with ICRE-03 followed by ICRE-025 (Table 2 and 3) at the two test sites with variable magnitude implying that the elite Rhizobia strains were performing at equal level with the standard check (EAL-029) even at higher magnitude. Generally chickpea varieties treated with rhizobial inoculant produced higher mean value of symbiotic traits and the current results are in agreement with previous reports of Tena et al , Beshir et al , Abdula  and Argaw  that were conducted on chickpea and other grain legumes in which inoculation increased parameters of symbiotic and agronomic traits.
Another unique result of this study regarding non nodulating genotype ICC-4819 was its superior performance in symbiotic trait measurements. The highest shoot dry weight (63.39 gm plant-1 at Debre Zeit) and the highest nitrogen concentration (0.46% at Debre Zeit, 0.65% at Wolayta Sodo) were also observed from straw sample of genotype ICC-4918. The possible explanation for high shoot dry weight and high nitrogen concentration in genotype ICC-4918 was its late maturity. In fact DTM for ICC-4918 was 97 days and 117 days at Debre Zeit and Wolayta Sodo respectively (table 2 and 3), that means it was late by 7 days from early maturing variety. According to Bidlack et al  findings, late maturing varieties accumulated more biomass in vegetative shoot components (leaf and stem) while early maturing varieties partitioned more photosynthate into reproductive structures (flower, pods, seeds).
Regarding compatibility and effectiveness of elite indigenous rhizobia in the current study, strains ICRE-03, IRCE-025 and ICRE-05 were found to be superior in most symbiotic parameters measured compared to the standard check (EAL-029). For instance comparison of rhizobia strains performance in measurements of shoot dry weight and relative effectiveness (Percentage of nitrogen increased), chickpea varieties inoculated with strain ICRE-03 had the highest shoot dry weight (56.46 gm plant-1) followed by strain ICRE-05 (52.35 gm plant-1) at Debre Zeit. Similarly in the experiment conducted at Wolayta Sodo varieties inoculated with strains ICRE-05 had the highest shoot dry weight (52.24 gm plant-1), followed by strain ICRE-025 (50.98 gm plant-1). Other comparison criteria for effectiveness of rhizobia in symbiotic nitrogen fixation is a parameter known as relative effectiveness (RE) as reported by Imran et al  and Maâtallah et al . Thus, chickpea varieties inoculated with strain EAL-029 had the highest (23.88%), relative effectiveness value followed by strain ICRE-03 (21.29%) at Debre Zeit. Similarly, under experiment conducted at Wolayta Sodo varieties inoculated with strain ICRE-025 gave the highest relative effectiveness (31.34%) followed by strain ICRE-03 having relative effectiveness value of 25.84% implying that all the elite rhizobia strains under the study were compatible and effective in symbiotic nitrogen fixation as long as biotic and abiotic factors were kept constant.