Journal of Microbial & Biochemical Technology

Screening for Ecologically Competent, Nutritional Characteristics and Symbiotically Effective Chickpea Nodulating Mesorhizobium spp. Isolated from Acidic Soils of Ethiopia

Atsede Muleta¹, Kassahun Tesfaye², Teklehaimanot Haileselassie², Douglas R. Cook³ and Fassil Assefa^{1 1}Department of Microbial Cellular and Molecular Biology, P.O. Box 1176, Ethiopia
²Department of Biotechnology, P.O Box 1176, Ethiopia
³Department of Plant Pathology, University of California Davis, One Shields Ave, Davis, CA, USA
^*Correspondence: Dr. Atsede Muleta, Department of Microbial Cellular and Molecular Biology, Ethiopia, Email:

Received: 20-Feb-2021 Published: 23-Aug-2021

Introduction

Soil acidity is one of the most important factors that affect nitrogen fixation and the production of leguminous crops, because it increases Aluminum (Al) and Manganese (Mn) toxicity and hampers Calcium (Ca) and Phosphorus (P) uptake by plant [1]. Jaiswal et al. [2] have reported that phosphorus deficiency and Aluminium (Al) toxicity in acid soils severely affect growth and symbiotic nitrogen fixation in legumes. These stresses limits the persistence and survival of rhizobia strains in the soil, and affect the exchange of molecular signals between rhizobia and their hosts, thus reducing nodulation. However, legumes and their specific endosymbiontic rhizobia exhibit varied responses to acidity and effective symbiosis on the host under acidic stress depends upon the strain and legume variety [3]. Thus, legume production can be improved with the selection of acid-tolerant varieties, effective and competitive strains of rhizobia and liming in acidic soil [4].

Phosphorus deficiency is one of the constraints of legume production in soil acidic conditions. It is interesting to note that rhizobia are one of the most effective phosphates solubilizing rhizobacteria that can improve phosphorus nutrition in the soil [5]. Therefore, acid-mediated P deficiency in acidic soils could be alleviated by inoculating phosphate solubilizing bacteria [6]. Thus, isolating and characterizing of symbiotically effective chickpea rhizobia with phosphate solubilization and other multiple growth properties from acidic soils is crucial for inocula production to enhance legume production [5,7].

Chickpea is one of the most important leguminous crops nodulated by specific group of rhizobia under the genus Mesorhizobium, to fix atmospheric nitrogen and improve soil fertility. It is estimated that chickpea fixes inorganic nitrogen with suitable rhizobial partner to utilizable form to plants to the tune of 90-180 kg/ha/ yr [8]; depending upon the host variety, symbiotic and ecological competence of Mesorhizobium strains [9,10]. Inoculation of chickpea with ecologically fit mesorhizobial strains improved nodulation, growth and yield components of chickpea varieties under adverse conditions [11], which is partly measured by their in vitro ability to utilize different carbon and nitrogen substrates, their inherent resistance to different antibiotics and tolerance to environmental factors such as acidity (pH), temperature and salinity (salt) [12].

According to [13], chickpea is one of the successful leguminous crops better adapted to grow and fix nitrogen in acidic soils. Some strains such as Mesorhizobium loti have shown a high degree of acid tolerance in laboratory media, being able to grow at pH values as low as 3.0 [13,14]. Imen et al. have demonstrated that Mesorhizobium can have a dual purpose of effective symbiotic association for nitrogen fixation and phosphate solubilization to enhance chickpea production in acidic soils. Mesorhizobia isolates having nitrogen fixing as well as high P solubilizing capability have great value for sustainable yield enhancement.

Ethiopia is the major chickpea producing country in Africa [15]. However, it is estimated that about 43% of the total cultivated land area is affected by soil acidity [16]. It has been reported that soil acidity mainly in the central and western parts of the country limits chickpea and other legume crops production [17,18]. Under the circumstances, nitrogen and phosphorus are deficient in most highland acidic soils of Ethiopia.

In Ethiopia, several studies were undertaken on eco-physiological and symbiotic properties of chickpea nodulating rhizobia [19-23]. Most of these studies identified (5-10%) chickpea rhizobia isolates combined tolerance to different in vitro stress conditions and nutritional versatility with symbiotic effectiveness comparable to Nitrogen fertilizer control plants. Other studies also showed effective symbiosis and P solubilization by rhizobia nodulating faba bean [24,25], and soybean [26] from acidic soils. However, there is still a dearth of information on the pattern of ecological competitiveness, symbiotic effectiveness and phosphate solublization of the different taxonomic groups of Mesorhizobium spp. from acidic soils of Ethiopia. Therefore this study was initiated to screen low pH tolerant, phosphate solubilizing, heterotrophically competent, and symbiotically effective strains from eight indigenous Mesorhizobium species under in vitro laboratory and greenhouse conditions.

Methodology

Sources of Mesorhizobium Isolates

The study included 81 mesorhizobial strains were retrieved from roots nodule of chickpeas, collected from acidic soils in central, western southern and northern parts of Ethiopia. They were isolated on Yeast extract agar media, genetically identified into the genus Mesorhizobium spp (Table 1) [27] and deposited in culture collection centers of the Plant Pathology Laboratory, University of California Davis and Department of Microbial, Cellular and Molecular Biology, Addis Ababa University.

Table 1: Mesorhizobium strains and origin of culture collection used in ecological adaptations and symbiotic characterization.

Screening for Low pH Tolerance and Phosphate Solubilization of Mesorhizobium Strains

All the strains were screened for acid tolerance on medium consists of 300 μM MgSO₄.7H2O, 300 μM CaCl₂, 100 μM Fe EDTA. 10 μM KCI, 1μM MnCl₂.4H₂O, 0.4 μM ZnSO4.7H₂O, 0.1 μM CuCl₂.2H₂O, 0.02 μM Na₂MoO₄.2H₂O, 0.02 μM Co(N0₃)2.6H₂O, 500 μM KH₂P0₄,500 μM K₂HPO₄, arabinose (5.0 g), galactose (5.0 g), (1.1 g) Na glutamate, biotin (0.1 mg), thiamine (1.0 mg), (0.005%) bromothymol blue, (15g) agar, and 1L distilled water and adjusted to pH 5 containing high Mn (1.0 mM) and Al (50 μM), and low P (5 μM) and Ca (50 μm) [28]. The isolates were also prescreened for P solubilization on Pikovskaya medium containing inorganic tri-calcium phosphate. The medium contained; g/l (glucose 10, yeast extract 0.5, Ca₃ (PO₄)₂ (5.0), (NH₄)2SO₄ (0.5), NaCl (0.2), KCl (0.2), MgSO₄.7H₂O (0.1), MnSO₄.2H₂O (0.002), FeSO₄.7H₂O (0.002), and agar 15. One hundred μl of the inocula (109 cfu/mL) were spotted on the medium and incubated at 28° for 7-10 days. Isolates that formed clear zones around the colony were considered as phosphate solubilizers and the solubilization index (SI) was calculated according to Edi-Premono et al., [29].

SI= Colony diameter + Halo zone diameter

Colony diameter

Eco-Physiological and Nutritional Characteristics of Selected Mesorhizobial Strains

All tests, except carbon and nitrogen utilization, were carried out on YEMA plates by inoculating with 10 μl of inoculums suspension (109 cfu/ml) and incubated at 28°C for 5 days against inoculated control unless stated otherwise. All tests were carried out in triplicates and results were recorded visually as ‘’+’’ for growth and ‘’- ‘’ absence of growth.

Salt, pH and Temperature Tolerance

Salt and low pH tolerance was determined on YEMA plates containing 1 to 5% (w/v) NaCl concentrations and the medium adjusted to pH (4 and 4.5), respectively [30]. Temperature tolerance was evaluated by inoculating them on YEMA plates under incubation temperatures (4, 10, 15, 20, 37, 40 and 45°C) [31].

Intrinsic Antibiotic and Heavy Metal Resistance

The intrinsic antibiotic and heavy metal resistance of strains were determined on solid YEMA medium containing filter sterilized antibiotics or heavy metals. The stock solutions of the antibiotics or heavy metals were sterilized using (0.22 μm Millipore) membrane filters and added to YEMA. The antibiotics used were (μg. mL^-1): ampicillin (5 and 10), chloramphenicol (5 and 10), erythromycin (10 and 20), nalidixic acid (5 and 10), streptomycin (40 and 80), rifampicin (5, 10), neomycin (5 and 10) and tetracycline (5 and10). Similarly, the heavy metals used were CoCl₂ (25,100), CuCl₂.2H₂O (50, 100), NiSO₄ (50,100), and ZnCl₂ (50,100), K₂Cr₂O₇ (50, 100) [32]. Acidity related Al³⁺ and Mn²⁺ tolerance was tested at two different Al concentrations KAl(SO₄)₃ (50 μM and 100 μM) and two Mn concentrations MnCl₂ (75 μM and 100 μM) using Keyser and Munns (KM) agar medium under acidic conditions at pH 5 [33].

Nutritional Versatility of Strains on Different Carbon and Nitrogen Substrates

Different carbohydrates were added as described by Amarger et al. [30] at a final concentration of 1gL^-1 of the basal medium containing (gl/1): K₂HPO₄, 1; KH₂PO₄, 1; FeCl₃.6H₂O, 0.01, NH₄SO₄, 1NaCl₂, 0.1; MgSO₄.7H₂O, 0.2; and agar, 15. The following filter sterilized (0.22 μm millipore) heat liable carbon sources; Citric acid, D-sorbitol, D-glucose, D-galactose, xylose, trehalose were added after autoclaving; and heat stable α-lactose, D-fructose, glycerol, α-cellulose, sucrose, and maltose were autoclaved with the basal medium. Filter sterilized L-tryptophan, methionine, L-tyrosine, leucine, riboflavin; DL-β-phenylalanine, L-arginine, glutamic acid, L-lysine, L-serine, glycine, and thiamine were used as a sole nitrogen source to a final concentration of 0.5 gL^-1 on the basal medium containing (gL^-1): K₂HPO₄, 1; KH₂PO₄, 1; FeCl₃. 6H₂O, 0.01; MgSO₄.7H₂O, 0.2; CaCl₂, 0.1; NH₄ (SO₄)₂, 1; and agar, 15; from which (NH₄)₂(SO₄) was omitted and mannitol was added after autoclaving [30]. All plates were incubated at 28°C for 5 days.

Symbiotic Characterization Mesorhizobia Strains

The experiment was carried out at Debre Zeit Agricultural Research Center (DZARC), Debre Zeit, Ethiopia. The study was undertaken in a pot experiment using a sterile sand culture under greenhouse condition. Each Mesorhizobium strain was grown on YEMB and incubated at 28°C for 5 days. Seeds of chickpea cultivars called ‘Natoli and DZ-ck-2011 s-2-0042’ were surface sterilized with 4% sodium hypochlorite for 3 min, then rinsed with five changes of sterile distilled water and allowed to germinate on water agar 1.5 % w/v at 250C for three days.

The germinated seeds were planted in alcohol swabbed plastic pots (3 kg capacity) containing washed and autoclaved sterilized river sand. Five seeds were planted pot-1, individually flooded with 1 ml of the culture suspension (10^-9 cfu/ml) and thinned down to three plants pot-1 after 5 days of emergence (DAE). The experiment was laid out with three replications for each treatment using randomized complete design with 12 h photoperiod, day temperature (28 ± 2⁰C) and night temperature (17 ± 3⁰C), by including uninoculated but nitrogen-fertilized (1% w/v KNO3) pots as positive (TN) control and uninoculated non- nitrogen-fertilized (T0) pots as negative controls. The pots were irrigated with nitrogen-free plant growth nutrient solution CRS (Center for Rhizobium Studies, Australia once a week and with sterile distilled water every three days, respectively. The pH of the nutrient solution was adjusted to pH5.

Plants were harvested after 8 weeks of planting to record the number of nodules plant^-1 (NN), nodule dry mass (NDW); shoot dry weight (SDW). The percent symbiotic effectiveness (SE) was calculated according to [34]. The percent symbiotic effectiveness of the isolates was expressed as a percentage of the shoot dry biomass of each treatment compared with the shoot dry biomass of the positive control (with N). Finally, symbiotic effectiveness was rated as highly effective (HE) when the percentage of effectiveness >80%, effective, (E) between 50 and 80%, and of low effectiveness (LE) between 35 and 50%. Strains were considered ineffective when the percentage effectiveness was less than 35%. The data were analyzed by one-way analysis of variance (ANOVA) using the general linear model procedure of the SAS software package (SAS/STAT; version 9.3) and mean values were separated according to Duncan’s multiple range test at p = 0.05.

Results and Discussion

Screening of Mesorhizobia Strains for Low pH Tolerance and Phosphate Solubilization

Chickpea nodulating Mesorhizobium strains were screened based on low pH tolerance and 62 (76%) selected strains showed growth on low pH 5 medium (data not shown), indicating the presence of low pH tolerant indigenous strains. Other studies also showed the presence of low-pH tolerant chickpea rhizobia in Portugal [13] and Morocco [31]. The data showed that the growth of isolated strains varied ranging from none to profuse; indicating the strains of a given species varies in their pH tolerance. Such differences in tolerance to acidity among strains have been reported previously for various Rhizobium [26,33,35] indicated that Rhizobium strains that survived in the acid soil cannot grow on a nutrient medium with a pH as low as that of the soil from which the strains were isolated. According to Icgen et al. [36], chickpea rhizobia strains displayed a tendency to neutralize the pH of the medium when grown freely in media adjusted to different low pH values, and this might account for the success in acidic conditions. Overall, the present work shows that in vitro evaluation of strain growth under pH stress may also be a useful method for finding rhizobial isolates adapted to different soil pH (Table 2).

Table 2: Eco-physiological characteristics of selected chickpea nodulating mesorhizobia strains grown YEMA medium and incubated for 5-7 days.

A large number of the mesorhizobia strains 47(76%) were capable of solubilizing insoluble inorganic phosphate sources (tri-calcium phosphate) with solubilization index (SI) ranging from 1.3 to 3.1 (Table 3) of which Mesorhizobium a.138w (ANI95 groups 2D) showed the largest solubilization index (SI = 3.2), followed by Mesorhizobium a.117L2 (ANI95 groups 2D) and a.71 (ANI95 groups 4B) (SI = 3.0). Previous studies in Ethiopia also proved that chickpea rhizobia have phosphate solubilizing characteristics with solubilizing efficiency (SI) of 0.5 to 1.3 [19,20]. This data showed the number of (population density) (76%) and the solubilization indices of the mesorhizobial strains isolated from acidic soils was higher than the ones previously reported (30-44%) from other relatively high pH soils in Ethiopia [19,20] and 70% in India. According to Peix et al., [5] chickpea rhizobia, are the best phosphate solubilizing of all root nodule bacteria on plates, better mobilize phosphate from TCP and significantly increase growth of the host plant.

Table 3: Soil acidity related metal (Al3+ and Mn2+ toxicity) tolerance at pH 5 of selected chickpea nodulating mesorhizobia strains.

Eco-Physiological Characteristics of Selected Mesorhizobium Strains

The mesorhizobial strains were prescreened for tolerance to pH 5, of which 26 strains were selected for further analysis. The strains were tested for in vitro tolerance at lower pH, 9 and 25 strains were grown at pH 4 and 4.5, respectively (Table 2). Studies also indicated that Mesorhizobium loti grown in medium at pH values as low as 4 [14] and other chickpea mesorhizobia isolates are able to grow at strongly acidic pH 3 [13]. The result indicated that tolerance to acidity is more strain specific than species specific [30]. Interestingly, a.15star (ANI95 groups 5C) showed growth at pH 5 but did not grow at pH (4, 4.5) and thus might be considered a moderate acidophile. Similarly, Brigido et al. [13] and Laranjo and Olivera, [12] have shown the isolates that belong to the group of M. ciceri with preference for tolerance at pH 5, which suggest that a species-related tolerance to acidity. The data did show a negative correlation (r=-0.09,-0.26, p=0.05) between origin of isolation soil pH and pH of isolation medium which is contrary to reports that show a positive correlation between chickpea rhizobia tolerance to different pH values and the origin-soil pH [13,30,38]. According to Brigado et al. [39], adaptation to acid pH by chickpea rhizobia is due to the presence of chaperones genes in their cells which are important for survival during acid stress.

All the strains were able to grow between 20 and 37°C and showed variations below and above these values, where 6(23%) strains were resistant to 10°C; whereas 11 strains (42.3%) were able to grow at 45°C (Table 2), which is relatively different from temperature sensitive chickpea rhizobia (40-45°C) isolated from Ethiopia [22] and China [10]. Conversely, studies from Turkey [40], and Morocco [31] showed chickpea rhizobia were able to grow at high temperature (>400C), which could be related to local adaptation. Mesorhizobium a.117L2 (ANI95 groups 2D) and a.200M (ANI95 groups 3A) strains showed wide range of temperature tolerance (10- 450C) than the other strains (Table 2). Similarly, Laranjo and Olivera [12] and Rai et al. [41] have shown pattern of tolerance to high temprature by Mesorhizobium plurifarium and Mesorhizobium loti strains, respectively. According to Rodrigues et al [37], adaptation of tolerance to high temperature by rhizobia strains in chickpea rhizobia is due to overproduction of a set of proteins, termed heat shock proteins (HSPs), which are important for survival during stress conditions.

Chickpea rhizobia strains displayed high diversity in their salt tolerance (Table 2). All strains were tolerant to 1% NaCl, but 24 (92%) of the strains survived at 2% NaCl, and fewer isolates 7 (27%) were able to grow on YEMA medium containing 5% NaCl. This result is comparable to previous finding in Ethiopia that indicates a wide range of variation in salt tolerance to 1-5% (w/v) NaCl concentration [21]. The most tolerant strains were a.AR1 (ANI95 groups 2E), a.30s (ANI95 groups 1C), a.66 (ANI95 groups 1C), a.161 (ANI95 groups 8A), a.452 (ANI95 groups 8A), a.55 (ANI95 groups 11A) and a.15star (ANI95 groups 5C) that were able to grow at 5% NaCl. Studies also showed that Mesorhizobium ciceris, Mesorhizobium plurifarium and Mesorhizobium loti strains were tolerant to high NaCl concentration [12,41]. According to Zahran [42] salt tolerant rhizobia are endowed with the capacity to tolerate osmotic stress that is mainly associated with their capacity to accumulate low molecular weight organic solutes in their cells.

Acidity-Al³⁺/Mn²⁺ Tolerance

The strains showed variable responses to Al3+ and Mn2+ toxicity which is often associated with acidic soils (Table 3). Thus, 7(27 %) and 10 (38%) of the Mesorhizobium strains displayed tolerance to Al3+ and Mn²⁺toxicity at a concentration of 100 μM /ml at pH 5, respectively. The data showed that strains a.117L2 (ANI₉₅ groups 2D), a.64 (ANI95 groups 2E), a.152 (ANI₉₅ groups11A), a.55 (ANI₉₅ groups 11A), a.200M (ANI₉₅ groups 3A) and a.71 (ANI₉₅ groups 4B) were the most tolerant strains that grew well at all tested concentration of Al³⁺ and Mn²⁺. The current result also confirmed the earlier report of Ayanaba et al. [33] who stated that, there is relationship between acid-Al sensitivity of isolates with their colony texture, as large-mucoid rhizobial colonies were more resistance than dry-pinpoint colonies. Previous studies in Ethiopia [19] and Morocoo [31] also showed relatively same pattern of resistance to Mn²⁺, and sensitivity to Al³⁺ in chickpea rhizobia isolates. According to Jaiswal et al. [2], Al³⁺ toxicity and acidity itself is probably more important limiters of rhizobial growth than Mn²⁺ toxicity in acid soils.

Intrinsic Antibiotics (IAR) and Heavy Metals Resistance (HR)

The Mesorhizobium strains showed variations in inherent antibiotic resistance in that almost all the strains were resistant to nalidixic acid, kanamycin, ampicillin, chloramphenicol and erythromycin with the exception of Mesorhizobium a.71 (ANI95 groups 4B) and a.89 (ANI95 groups 4B) that were sensitive to erythromycin (Table 4). Three Mesorhizobium strains, a.71 (ANI₉₅ groups 4B), a.55 (ANI₉₅ groups 11A) and a. AR7 (ANI₉₅ groups 1D) were resistant to streptomycin and a. AR7 strain from M. genospeces 1D group were resistant to tetracycline. Mesorhizobium a.15star (ANI95 groups 5C) and a.AR7 (ANI95 groups1D) showed multiple antibiotic resistances by growing on media containing 7-8 of the antibiotics tested (Table 5). This is similar to a previous report that showed chickpea rhizobia such as M. ciceri strains tolerant to 70-80%) tested antibiotics [23]. Other studies in Ethiopia also showed that chickpea rhizobia were relatively resistant to nalidixic acid and erythromycin; and sensitive to streptomycin and tetracycline [21]. On the contrary, chickpea rhizobia from Morocoo [31] and Turkey [40] were sensitive to ampicillin, chloramphenicol and kanamycin, indicating large variability in antibiotic resistance of chickpea endosymbionts.

Table 4: Intrinsic Antibiotic resistance (IAR), Heavy metals tolerance of selected chickpea nodulating mesorhizobia strains.

The mesorhizobial strains showed different response to heavy metals (Table 4). Accordingly, all strains failed to grow on Co and Ni (data not shown), whereas most strains (65-96%) were tolerant to aluminum, zinc, manganese, copper and chromium. The data showed that 12 (46) % of the strains showed a wide spectrum of heavy metal resistance growing on five of the heavy metals tested. The study in Ethiopia also showed that chickpea rhizobia were resistant to Mn, Al, and Zn. A study from Morocco also showed 20-60% of the chickpea rhizobia were resistant to Al, Zn, and Cu. This generally indicates that there is inherent antibiotic and heavy metal resistance by strains based upon the origin of isolation and the type of chemicals they had been exposed to in the soil.

Pattern of Carbon and Nitrogen Source Utilization

Most of the chickpea mesorhizobia strains were able to utilize many of the carbon substrates tested and failed to grow on citric acid (Figure 1). Mesorhizobium a.15star (ANI95 groups 5C) and a.AR452 (ANI95 groups 2D) strains were the most versatile of all the strains that were able to grow on more than 90% of the carbon sources; whereas Mesorhizobium strains a.71, a.89 and a.8star (ANI95 groups 4B) group limited to grow on 50% of the tested carbon substrates. This result is concurrent with findings of Sirag and Assefa [23] that showed most M. plurifarium and M. ciceri strains from Ethiopia were versatile in carbohydrate utilization than the other groups.

Figure 1. Carbon and nitrogen utilization pattern of selected chickpea nodulating mesorhizobia strains
A. Carbon Utilization, B. Nitrogen Utilization.

Chickpea rhizobia strains also exhibited diversity in utilizing different amino acids and vitamins as sole N-sources (Figure 1). All strains were grown on all the tested N-sources whereas fewer strains failed to grow on L-lysine and glycine. Unlike that of carbon sources, 14 (54%) of the strains utilized more than 90% of the nitrogen substrates, whereas a.8star (ANI95 groups 4B) strain utilized only 33% of the tested N sources substrates, indicating that most Mesorhizobium strains were more versatile in N utilization than C utilization. Other studies in Ethiopia also showed the same pattern of carbon and nitrogen utilization of chickpea rhizobia. On the contrary, some M. ciceri and M. plurifarium strains utilized citrate and lysine unlike to the present finding.

This implies that chickpea rhizobia display large variability in carbon and nitrogen substrate utilization that may be related to differences in strains/genotype or local adaptations [43, 44].

Symbiotic Effectiveness of Mesorhizobium Strains

The greenhouse trial of selected strains on a sand culture under low pH 5 showed considerable variations among the tested mesorhizobial strains in shoot dry mass, nodule number, nodule dry mass per plant and symbiotic effectiveness (SE) compared to the control (p ≤ 0.01) (Table 5). The inoculated strains induced nodules on both chickpea varieties ranging from 10 NN/plant with Mesorhizobium a.AR1 (M. genospecies ANI₉₅ groups 2E) to 98 NN/ plant with Mesorhizobium a.64 (ANI95 groups 2E) (mean= 61 NN/ plant) on Natoli variety; and from 12.5 NN/plant for Mesorhizobium a.90 (ANI95 groups 10A) to 85 NN/plant with Mesorhizobium a.89 (ANI₉₅ groups 4B) (mean= 38 NN/plant) on DZ-ck-2011 s-2-0042 variety.

Table 5: Symbiotic characteristics of chickpea nodulating mesorhizobia strains at pH 5.

The nodule dry mass was within the range of 49 mg/plant and 119 mg/ plant (mean=80 mg/ plant) with Natoli variety, and 57 to 102 mg/plant (mean=77 mg/plant) on DZ-ck-2011 s-2-0042 host variety, respectively (Table 5). The strains also induced the accumulation of shoot dry weight ranging from 0.63 g/plant to 1.53 g/plant (mean= 0.98 g/plant) on Natoli variety; and 0.66 g/ plant to 1.22 g/plant (mean= 0.85 g/plant) on DZ-ck-2011 s-2-0042 variety (Table 5).

This study indicated that the number of nodule and dry mass under low pH condition was much lower than recorded in other studies which agreed with reports from [45,46]. The average nodule dry mass obtained from both varieties were also much lower than 120 mg/per plant reported by Jida and Assefa [19], 200 mg/plant by Tena et al. [21] and 212 mg/per plant from chickpea which was reported by Brigido from moderately acidophilic mesorhizobia. The low nodule dry mass could also be attributed to direct pH stress on the plant, limiting nutrient uptake and subsequent dry matter accumulation [42].

On the basis of relative shoot dry matter accumulation in reference to N fixing and control plants, 5 (24%) strains were highly effective (HE) and 10 (48%) strains were effective (E) on both varieties. However, all the Mesorhizobium strains were highly effective (HE) and effective (E) on DZ-ck-2011s-2-0042 variety, but only 24% and 48 % of these strains were highly effective (HE) and effective (E), respectively on Natoli variety; and Mesorhizobium strains, a.55 (ANI₉₅ groups 11A), a.AR7 (ANI95 groups 1D), a.66 (ANI₉₅ groups 1C), a.152 (ANI95 groups 11 A), a.AR1 (ANI₉₅ groups 2E) and a.222 (ANI₉₅ groups 1D) strains were lowly effective on Natoli variety, indicating variations in their symbiotic compatibility and interaction between rhizobia and the host genotypes [11,46,47]. In all cases, the Natoli variety showed higher nodule number per plant, nodule dry weight, shoot dry weight, but not higher values in symbiotic effectiveness than the DZ-2012-CK-2011s-2-0042 variety. Alemu and Lule [18] have indicated that chickpea genotypes showed differential response to acidic pH condition.

In general, combined evaluation of ecological competitiveness (in vitro) and symbiotic effectiveness data showed that five strains namely; a.117L2 (ANI₉₅ groups 2D), a.15star (ANI₉₅ groups 5C), a.71 (ANI₉₅ groups 4B), a.40L2 (ANI₉₅ groups 8A) and a.200M (ANI₉₅ groups 3A) performed better than the other strains, and even out-performed over the commercially available local strain Cp41 on both plant varieties at pH 5 under greenhouse conditions. This is concurrent with a report that M. ciceri strains from Portugal were effective strains with (SE) > 75%, whereas M. ciceri and M. plurifarium strains from Ethiopia were highly effective on both varieties.

In this experiment, the most highly effective strains were obtained from pH 5 condition compared to other investigator reports on acidic soil in Ethiopia. Kenasa et al. [24] have revealed that rhizobial isolates of faba bean collected from acidic soils of Wollega, western Ethiopia were effective on faba bean, whereas, Muleta et al. [26] have demonstrated that rhizobia strains of soybean collected from acidic soils of Ethiopia were effective from which, only 4% of the soya bean strains were found to be highly effective. It is likely that symbiotic performance of the strains is significantly dependent on pH condition. Thus, this result underlines the importance for a local screening of symbiotically effective and ecologically competent mesorhizobia isolates from acidic soil to enhance grain yield of the chickpea crop in acidic soil.

Conclusion

In the present study it can be concluded that Ethiopian acidic soils harbored symbiotically effective chickpea nodulating Mesorhizobium spp. These strains were also ecologically competent and heterotrophic versatile in carbon and nitrogen utilization, with a wide range of in vitro tolerance to various stress conditions, including low pH, Mn²⁺ and Al³⁺ toxicity, salinity, high temperature, heavy metals and antibiotics indicating their potential to effectively nodulate and fix nitrogen under field conditions.

The strains showed variations in their symbiotic effectiveness in nitrogen fixation on the two host varieties showing better performance on DZ-ck-2011s-2-0042 variety. Five strains: a.15star (ANI₉₅ groups 5C), a.117L2 (ANI₉₅ groups 2D), a.71 (ANI₉₅ groups 4B) and a.40L2 (ANI₉₅ groups 8A), and a.200M (ANI₉₅ groups 3A) were relatively superior in their symbiotic performance, compatibility with both chickpea varieties, and can be recommended as prospective commercial inoculants provided they can be tested in field trials in acidic soils.

Funding

This work was supported by USAID-PEER Program Cycle-4, administered by the U.S. National Academies of Sciences, Engineering, and Medicine (NASEM).

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

The authors would like to thank the Department of Microbial, Cellular and Molecular Biology, Addis Ababa University, Department of Plant Pathology, University of California Davis, USA and Debre Zeit Agricultural Research Center are acknowledged for their supports.

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