Isolation and Characterization of Chickpea ( Cicer arietinum L.) Nodulating Rhizobia Collected from South Wollo Zone, Ethiopia

This study aimed at screening rhizobial isolates of chickpea and evaluating their symbiotic nitrogen ﬁxation eﬃciency and tolerance to abiotic stresses. A total of 107 (100%) isolates were collected of which 52 (48.6%) were conﬁrmed as chickpea rhizobia using preliminary tests. Among 52 (48.6%) isolates, 46 (88.5%) have induced nodulation on chickpea under greenhouse and were screened under in vitro conditions and 6 (11.5%) of them were discarded due to fail to nodulate. The greenhouse data showed the highest nodule number (68.67 plant − 1 ), nodule dry weight (0.17g plant − 1 ), and shoot dry weight (0.81 g plant − 1 ) were scored by plants inoculated with isolates WUCR 17, WUCR 1, and WUCR 66, respectively. Among authenticated isolates, 73.9%, 21.7%, and 4.3% were highly eﬀective, eﬀective, and lowly eﬀective, respectively. The physiological test showed 15% of isolates tolerated 13% salt concentration and 10.9% of isolates grew at pH 4–10 range. All isolates grew at 20–35 ° C and 13% grew at maximum temperature (50 ° C). The isolates showed better resistance to the tested antibiotics at low concentration (2.5 μ g/ml) but the majorities were sensitive at high concentration (10 μ g/ml). Among the isolates, 13% tolerated all tested heavy metals but 48% were sensitive to mercury. Regarding the carbohydrate utilization test, 52.2% catabolized all the tested 11 carbon sources but 6.5% of them utilized only 63.6% carbon sources. Amino acid utilization showed isolates (85%) utilized D-alanine except WUCR (14, 25, 31, 34, 39, 59, and 76) and 76% of the isolates utilized arginine and phenylalanine and 74% utilized leucine as a nitrogen source. Of the isolates, 43.5% of them utilized both D-alanine and arginine as a nitrogen source. In almost all tests conducted, isolates WUCR 1 and 5 performed top and were recommended as potential candidates for microbial inoculants.


Introduction
Chickpea (Cicer arietinum L.), a member of the family Leguminosae, is an important pulse crop valued as a rich source of proteins [1].It is a cool-season legume crop that is grown in many countries around the world as a food source [2].It has an important role in the maintenance of soil fertility, particularly in dry, rain-fed areas, and can resist drought [3].Chickpea is a staple basic food crop in many tropical and subtropical Afro-Asian countries and is one of the world's major pulse crops which are traditionally cultivated in marginal areas and semiarid regions [4].Globally, it ranked the third most important pulse crop [5] with an average yield of 913 kg/ha and a total production of 10.9 million metric tons [6].At present, it is produced in over 40 countries of the world of which 89.7% is in Asia, followed by 4.3% in Africa, 2.6% in Oceania, 2.9% in Americans, and 0.4% in Europe [7].About 95% of chickpea cultivation and consumption is in developing countries [8].
Ethiopia is the first country in chickpea production and production area coverage in Africa [9] where it plays an important role in the local economy and contributes to sustainable agriculture.Currently, chickpea is cultivated in four regions of the country: Amhara, Oromia, Southern Nations Nationalities and People's Region (SNNPR), and Tigray.Amhara and Oromia regions together produce 93% of total chickpea production in Ethiopia while SNNPR and Tigray produce 3.5% and 3%, respectively [8]. e top 9 chickpea producing administrative zones (North Gonder, South Gonder, North Shoa, East Gojam, South Wollo, North Wollo, West Gojam, and Gonder Zuria) of the Amhara region account for about 80% of the country chickpea production [10].However, its yield has remained very low compared to the natural potential of the crop due to several environmental stresses which prevail under Ethiopian edaphoclimatic conditions [11,12].e importance of selection, commercialization, and application of rhizobia (inoculants) with host pulse crops has been well known for decades [13].Microbial inoculants are the promising components for integral solutions to agroenvironmental problems because they promote plant growth by enhancing nutrient availability and uptake as well as maintaining the health of the plant [14]. is requires screening compatible hosts, endosymbionts, and appropriate environmental conditions.e inoculation of chickpea seeds with rhizobium is known to increase nodulation, nitrogen uptake, growth, and yield parameters of legume crops and some other biocontrol characteristics [15].Biofertilization has great importance in decreasing environmental pollution and deterioration of nature [15].Poor nodulation and the lack of inoculation in field experiments have frequently been reported worldwide, raising doubts about the efficiency of crop inoculation [16][17][18].e lack of response to inoculation can be attributed to intrinsic characteristics of both the host plant and the bacteria as well as the great sensitivity of the symbiosis to environmental stresses such as high temperatures, soil dryness, and low soil fertility [19,20].
ere are severe problems in soil fertility and crop productions due to excessive use of inorganic fertilizers causing undesirable effects on agriculture, food, biodiversity, and the environment at large [21,22].In Ethiopia, low soil fertility and limited external inputs are the major constraints to the low yield of crops [23] particularly low levels of nitrogen and available phosphorus in the country [24].In agriculture, perhaps 80% of the biologically fixed nitrogen comes from symbiosis involving leguminous plants and bacteria of the family Rhizobiaceae.Nitrogen, a vitally important plant nutrient, is the main factor that limits chickpea production in Ethiopia.However, these legumes form an effective symbiotic association with a soil-dwelling bacteria known as rhizobia and fix atmospheric nitrogen via biological nitrogen fixation (BNF) process in a plant root structure (nodules).e nitrogen fixed via BNF is mostly used for plant growth and development.is accounts for about 60% of the global nitrogen budget and is eco-friendly and cost-effective [25].e symbiotic association that forms BNF is affected by various factors [25] such as the rhizobium strains, host legumes, and biotic and abiotic soil factors.
Researches that focused on the diversity and symbiotic properties of rhizobia and rhizobacteria associated with pulse crops including fava bean, field pea, chickpea, and lentil were carried out [26,27].ese studies mainly focused on isolation and in vitro characterization under laboratory conditions and nitrogen fixation under greenhouse conditions [10].ese trials were limited to only a few areas of the country and no literature report in the study area so far [28][29][30][31][32]. e plant growth promoting and controlling role of chickpea rhizobia was forgotten except few works done in other areas outside from the study area and on other crop legumes [33,34].us, this study was aimed at isolating and characterization of chickpea (Cicer arietinum L.) nodulating rhizobia collected from South Wollo Zone, Ethiopia.

Sampling Sites and Sample Collection.
e sampling sites were comprised of 40 sampling sites from 4 woredas of South Wollo Zone including Tehuledere, Kutaber, Dessie Zuria, and Kalu (Figure 1), which were selected based on the high chickpea production history and no inoculation of chickpea rhizobia before.e root nodules were collected between October and November 2019 during flowering stage.Pink-colored nodules and 3 kg of soil were collected from the roots and rhizosphere of a healthy standing plant, respectively.e nodules were collected and placed in sealed vials containing a desiccant (silica gel) covered with 1 cm of cotton wool and brought to Wollo University Biology Laboratory and kept at 4 °C till isolation of the rhizobia was conducted [35].e soil was placed in polythene plastic bags and brought to the laboratory for further work.

Isolation of Rhizobia and Purification of the Isolates.
Dehydrated or desiccated root nodules were immersed in sterile distilled water overnight in labeled sets of Petri dishes [36].e imbibed nodules were surface-sterilized according to [36].e surface-sterilized nodules were transferred into sterile Petri dishes, crushed, and streaked on Yeast Extract Mannitol Agar containing Congo red (YEMA-CR).e plates were incubated at 28 ± 2 °C for 3-5 days and presence and absence of growth were recorded (36).

Presumptive Test.
e growth of the rhizobial isolates was determined by growing them on Peptone Glucose Agar (PGA), Keto-Lactose Test, Gram staining, and YEMA-CR medium [18,36,37].e rhizobia isolates were tested for determining their Gram reaction type using the KOH method [33,38].e isolates were incubated at 28 ± 2 °C for 3-5 days to check the presence and absence of growth.Isolates that showed no growth or very poor growth on glucose peptone agar medium were taken as rhizobia.e media composition includes (g/l) peptic digest of animal tissue (20), dextrose (10), sodium chloride (5), and agar (15).

Colony and Growth Characteristics.
e morphological characteristics of each isolate were determined according to [39][40][41].e isolates were inoculated onto YEMA and incubated at 28 ± 20 °C for 3-5 days to determine colony characteristics [40].Acid-base production was also determined [41].eir growth was assessed on YEM broth in an Erlenmeyer flask incubated in a Gyratory shaker at 130 revs.min − 1 and their doubling time was calculated from the logarithmic phase [42] as 2 International Journal of Agronomy where t is the time elapsed, Xo is the first OD reading, and X is the second OD reading in the logarithmic phase.

Greenhouse Authentication of Rhizobia.
Pots were washed and surfaced-sterilized and autoclaved as indicated in Somasegaren and Hoben [35].Chickpea variety called "Desi" was used for the greenhouse experiment.Gray undamaged seeds of uniform size were selected and surface-sterilized and were rinsed in five changes of sterile water and placed to 0.75 (w/v) water agar plates.ey were incubated at 28 ± 2 °C for 3 days.After three days, five germinated seeds were transferred into each surface-sterilized pot for germination which was thin down into three later.After 3 days, each seedling was inoculated with 1 ml of 72 hrs YEMB grown culture.e experiment was statistically laid out with three replications using a completely random design in a greenhouse with a 12 hrs photoperiod and an average of 28 °C and 15 °C day and night temperature.e experiment consisted of negative control, nitrogen-fertilization treatment, and bacterial treated treatments.All the plants were fertilized with quarter strength of Broughton and Dilworth N Free medium per week as described in Somasegaren and Hoben [35].All the pots were watered every two days.

Carbohydrate and Amino Acid Utilization.
e carbohydrate utilization test of the rhizobial isolates was evaluated using 11 carbon sources: D-fructose, mannitol, sucrose, lactose, and D-glucose, which are heat-resistant carbohydrates, and galactose, maltose, gelatin, cellulose, and starch, which are heat liable carbon sources, and from sugar alcohol, glycerol was tested [35].e two different types of amino International Journal of Agronomy acids which included D-alanine and arginine were used to determine the ability of the isolates to utilize amino acids as indicated in Amarger et al. [51].e plates were incubated at 28 ± 2 °C for 3-5 days and growth was recorded.

Data Analysis.
e data were analyzed and interpreted using one-way ANOVA.e experimental treatments were compared and contrasted against their control following Duncan's multiple range test (DMRT).
e correlation between different parameters was evaluated by using the Pearson correlation coefficient using SPSS version 20 and the dendrogram was constructed using the 65 phenotypic parameters used in the physiological test.
e phenotypic parameters were recorded as (1) for the presence of growth and (0) for the absence of growth during the test.

Isolation, Presumptive Test, and Cultural Characterization of Rhizobial
Isolates.Among a total of 107 (100%) chickpea nodulating rhizobial isolates, 52 (48.6%) of them were tentatively confirmed and 55 (51.4%) failed during preliminary test by conducting YEMA-CR absorption, growth on Peptone-Glucose Agar, and Keto-Lactose media and by their Gram reaction.Authentication work showed that 46 (88.5%) out of the 52 (48.6%) isolates form nodules and were authenticated as chickpea root nodule rhizobia.ese isolates were purified and designated as WUCR representing different numbers.e remaining 6 (11.5%) isolates failed to nodulate and were then discarded.Among the 46 isolates, 37 (80.4%) of them changed the YEMA-BTB medium into yellow color and the remaining 19.6% (WUCR 6, 11, 23, 28, 34, 55, 56, 61, and 82) changed the YEMA-BTB medium into blue which are characteristics of acid and base producing rhizobia.e colony morphology and the cultural characteristics of the isolates showed that 18 (39.1%) of them were large mucoid colonies (LM), 7 (15.2%)large watery (LW), and 21 (45.7%)large circular with extracellular exopolysaccharide production.Regarding the diameter of the isolates, all of them recorded colony diameter ranging in 2.0-6.0 mm. e largest colony diameter of 6.0 was displayed by isolates WUCR 6, 14, 28, and 106, whereas the smallest diameter of 2.0 mm was exhibited by isolates WUCR 2, 31, 33, 61, 82, and 89.e doubling time of the isolates ranged in 1.02-5.14hrs indicating that all the isolates were fast growers.Generally, the Gram reaction, presumptive test result, the colony morphology, cultural characteristics, and the generation time all showed characteristics of rhizobia and, therefore, the isolates could be tentatively characterized as chickpea rhizobia.

Symbiotic Effectiveness of Rhizobia Nodulating Chickpea under Greenhouse Conditions.
e shoot length, nodule number, nodule dry weight, shoot dry weight, and symbiotic effectiveness data obtained from the greenhouse experiment were presented (Table 1 3).
A low, moderate, and strong positive correlation were recorded among the symbiotic parameters at a significant level of 0.01 and 0.05 (Table 2).e correlation recorded between shoot dry weight and symbiotic effectiveness (r � 0.902) was strongly positive.

Pattern of Physiological Tolerance of Isolates.
Variation among isolates in their physiological tolerance to pH, temperature, salinity, antibiotics, heavy metals, and nutrient utilization to carbohydrate and amino acid sources was observed.

Tolerance to Salt, pH, and Temperature.
e isolates showed tolerance and sensitivity to the tested salt concentrations (Figure 4).All the isolates were tolerant to the salt concentration of 0.5% but showed a steady decrease in growth when they were inoculated into the medium containing 6 to 13% salt concentration.Consequently, 70% of the isolates grew at 7% NaCl, whereas 20% of isolates were resistant to a salt concentration of 12%.Seven (15.2%) of the isolates including WUCR 1, 3, 5, 11, 18, 29, and 31 grew at salt concentration of 13%.

Resistance and Sensitivity to Antibiotics and Heavy
Metals.
e isolates showed resistance and sensitivity to different antibiotics at different concentrations (Figure 7).e isolates were sensitive (58%) and resistant (98%) to chloramphenicol at 10 μg/ml and 2.5 μg/ml concentrations, respectively.irteen (28%) of the isolates were highly resistant to different antibiotics at different concentrations.e isolates showed variation in the tolerance and sensitivity to different antibiotics at different levels of concentrations.
e majority of isolates utilized the four tested amino acid substrates as sources of nitrogen (Figure 10).Most of the isolates (85%) utilized D-alanine except WUCR 14,25,31,34,39,59, and 76, and 76% of the isolates utilized arginine and phenylalanine and 74% utilized leucine as a nitrogen source.Of the isolates, 43.5% utilized both D-alanine and arginine.
e last cluster (C-IV) contained 3 isolates that are divided into two groups (IVA and IVB) with WUCR 2 and WUCR 1 isolates, respectively.Clustering decreased below 5% level of dissimilarity indicating their phenotypic diversity.

Discussion
In this study, among 107 root nodule bacteria collected from chickpea, 52 (48.6%) of them did not absorb Congo red, did not grow on PGA media, and were Gram-negative, which were characteristic features of rhizobia.
is result was similar to earlier works reported by Daniel Muleta [30] and Jida and Assefa [27] implying the isolates could be chickpea rhizobia.
e isolates changed YEMA-BTB into yellow (80.4%) and blue (19.6%) which was similarly reported by Jordan [41] and Lupwayi and Haque [39] indicating that they were fast (acid producers) and slow (base producers) growing rhizobia [52].Regarding growth rate, 10 (22%), 29 (63%), and 7 (15%) showed doubling time of <2 hrs, 2-4 hrs, and 4-5.14 hrs, respectively, which was in line with the findings of Jida and Assefa [27] who reported doubling time of fewer than 6.0 hrs.e isolates are large mucoid (39.1%), large watery (15.2%), and large circular (15.2 with extracellular exopolysaccharide production as described by earlier authors [27,32,34,[53][54][55].Moreover, the isolates exhibited a colony diameter ranging in 2-6 mm which was similarly reported [53,54].All the presumptive tests, colony, and cultural characteristics were characteristics of rhizobia indicating that the isolates can be categorized as chickpea rhizobia and previous studies in other locations have established that most chickpea rhizobia belong to M. ciceri or M. mediterraneum [52,56]. Among the isolates subjected to greenhouse authentication, 46 (89%) were authenticated as root nodule rhizobia, whereas 6 (11%) failed to nodulate which showed better nodulation compared to Daniel Muleta [30] who reported 47% nodulation failure that can be related to the inherent links    International Journal of Agronomy between the rhizobia and the hosts' capacity to fix nitrogen [57].e highest nodule number (68 plant − 1 ) was scored from isolate WUCR 17 and the least number (15 plant − 1 ) from isolate WUCR 37 which is nearly similar to the findings of Tadele Ereso [32] and Mulisa Jida and Fassil Assefa [27] who reported a maximum and minimum of 66, 79, and 7.5, and 41 nodule numbers per plant, respectively.Maatallah et al. [1] similarly reported nodule numbers ranging from 11 to 62 indicating agroecological zone is a major factor for rhizobia nodulation and nitrogen fixation.Plants inoculated by WUCR 1 and WUCR 14 produced maximum and minimum nodule dry weight of 0.17 g/p and 0.070 g/p which was less as compared to the result of Jida and Assefa [27] who reported 1.16 g/ p and 0.56 g/p as maximum and minimum nodule dry weight, respectively.e highest shoot dry weight (0.81 g plant − 1 ) was scored from plants inoculated by isolate WUCR 66 which was greater than the nitrogen-fertilization treatment (0.583 plant − 1 ) whereas the least mean shoot dry weight (0.277 g plant − 1 ) was obtained from plant inoculated with WUCR 37 and WUCR 101 which was greater than the negative control (0.240 g plant − 1 ).e result of shoot dry weight of this study was still less than the result reported by Jida and Assefa [27] that accumulated shoot dry weight of 0.60 g/p and 1.36 g/p indicating variation in the nitrogen-fixing capacity which could be due to difference in rhizobial strain type and other soil factors [58].e nodule dry weight directly correlated positively with the shoot dry weight which is a good indicator in determining factors of nitrogen fixation [55,59].e isolates showed symbiotic effectiveness ranging in 47.6-152.1% of which 73.9%, 21.7%, and 4.3% were highly effective, effective, and low effective, respectively.Jida and Assefa [27] reported 11% highly effective chickpea rhizobia.Similarly, Jida and Assefa [26] and Jida and Assefa [60] reported 33% and 24% highly effective rhizobia isolated from lentil and faba bean, respectively.e strong positive correlation between shoot dry weight and symbiotic   International Journal of Agronomy effectiveness (r � 0.902) showed more linear relationships than other parameters indicating that shoot dry weight is a good indicator of symbiotic effectiveness which is an important parameter for determining the symbiotic effectiveness of legume nodulating rhizobia [59].
In this study, all the isolates tolerated the tested pH at a range of 5-10 which was slightly similar to [54,61] which reported a pH range of 4.5 to 8.5 and [62] pH 6-10 implying that the isolates of this study grew at both lower and higher pH [1,53,63] which is an indication that chickpea rhizobia exhibit moderately acidic and alkaline characteristics.e isolates tolerated higher concentrations of salt (0.5-13%) which is not reported by earlier works [1,64] indicating that the isolates can be applied in areas of high salinity.Although there is variation, the isolates showed growth at both lower and higher temperature ranges (5-50 °C) which is closely similar to the findings of [1,65,66] indicating that the isolates can be applied in areas of low and high temperature [25].
e variation in the tolerance and sensitivity of the isolates to different concentrations could help for better coexistence with antibiotic-producing soil microorganisms in the soil.e occurrence of resistant isolates to the tested heavy metals could be important to improve crop yield when inoculated in soils contaminated with toxic heavy metals [63].All of the isolates catabolized a large variety of carbon sources that agreed with [27] which described that rhizobia catabolize a wide range of substrates and could have an ecological advantage which is a property of fast-growing rhizobia [41].e isolates utilized four different amino acid substrates as sources of nitrogen that agreed with [64,67] which reported most of the nitrogen sources were utilized by rhizobia nodulating chickpea.is utilization of carbon and nitrogen sources could be used as diagnostic features for the identification of the rhizobia and is important to improve their survival advantage [68].
e numerical analysis conducted based on 65 phenotypic traits grouped the isolates into four main clusters with several subclusters at 25% dissimilarity level (Figure 11).Clusters I, II, III, and IV comprised 26, 1, 16, and 3 isolates, respectively.Clustering decreased below 5% level of  e dendrogram constructed grouped the study isolates into different clusters indicating their ecological diversity and relatedness [55].In this study, chickpea rhizobial isolates endowed with multiple plant growth promoting and biocontrolling properties which could be applied as field conditions were screened.

Figure 1 :
Figure 1: Map of the study area (using Garmin GPS 72H).

Figure 3 :
Figure 3: Effect of rhizobia on shoot growth.

Figure 11 :
Figure 11: A Dendrogram highlighting the phenotypic diversity of chickpea rhizobial isolates.

Table 1 :
Performance of selected rhizobia isolates on potted sterilized sand under greenhouse conditions.

Table 2 :
Pearson correlation coefficient comparisons for nodule number, nodule dry weight, shoot dry weight, shoots length, and symbiotic effectiveness.Correlation is significant at the 0.01 level (2-tailed).* Correlation is significant at the 0.05 level (2-tailed).NN/p � nodule number per plant, NDW/p � nodule dry weight, SDW/P�shoot dry weight per plant, SL/ p � shoot length per plant, SE � symbiotic effectiveness.