Journal of Plant Pathology & Microbiology

Morphological Variation and Basic Characteristics of Selected Indigenous Rhizobia Isolated from Major Chickpea (Cicer arietinum L.) Growing in Regions of Ethiopia

Zehara Mohammed Damtew^* Ethiopian Institute of Agricultural Research, Debre Zeit Research Center, P.O. Box 2003, Addis Ababa, Ethiopia
^*Correspondence: Zehara Mohammed Damtew, Ethiopian Institute of Agricultural Research, Debre Zeit Research Center, P.O. Box 2003, Addis Ababa, Ethiopia, Email:

Received: 10-Sep-2021 Published: 27-Nov-2021

Introduction

Chickpea is one of the most important pulse crops used for food and feed in more than 60 countries worldwide mostly in the semi-arid tropics of sub-Saharan Africa and South Asia [1] and contributes to more than 20% of world pulse production [2]. It is integrated in soil cropping systems in the tropics for soil fertility management, for it fixes nitrogen with root nodule bacteria of the genus Mesorhizobium [3]. It is estimated that chickpea can produce up to 176 kg N ha-1 annually depending on cultivar, bacterial strain, and environmental factors [4]. Although chickpea is considered for long as very host specific to M. ciceri and M. mediterraneum [5].

The Mesorhizobium spp are saprophytic soil microbiota that have to persist and compete with other microorganisms to survive in the soil so as to effectively nodulate their host [6]. Their survival in the soil and their symbiotic performance are influenced by various abiotic factors temperature, soil pH, salinity, nutrients and environmental pollutants such as heavy metals and antibiotic produced by other microorganisms in the soil [7]. Stress tolerance tests and substrate utilization capability in vitro characteristics of rhizobial isolates is vital to obtain information about the adaptation to the intended soil environment of the Rhizobia in the organism’s habitat that may beneficially influence plant growth and development of the host plant [8].

This necessitates the primary screening of the isolates to test their tolerance to abiotic stresses such as acidity (pH), salinity (salt) and temperature in the laboratory conditions. Apart from abiotic factors, carbon and nitrogen utilization also showed their in vitro ability to metabolize à wide range of respiratory substrates. That will give a more-clear picture on environmental fitness of isolates in the soil for establishing N2-fixing symbiosis under adverse conditions.

Materials and Methods

Source of isolates and growth conditions

Fifteen test isolates were obtained as part of an earlier study by Greenlon A, et al. [9] and Damtew Z [10] and presented in Table 1. The isolates were kept in 20% (v/v) glycerol stocks at-80°C and retrieved on Yeast Extract Agar (YEMA) Medium at 28°C for 4-5 days for this characterization. Each isolate was characterized by colony appearance, diameter, color and extracellular polysaccharide production. The colonies were characterized as small creamy mucoid, large mucoid and watery less creamy mucoid. Growth rate were assessed by inoculating 1 ml of active rhizobial culture approximately (~109 cells/ml) in 10 ml of YM broth in flask and incubated on shaker for at least 4 to 5 days [11].

Then optical density of each respective isolate was measured at 540 nm using spectrophotometer starting at the time of inoculation and every 6 hours interval. After incubation viable colony forming units were determined through serial dilutions of the culture in sterile water and spread 0.1 ml of each dilution on YMA media in triplicate plate using sterilized glass. Plates contained 20-200 number of colonies were preferred to calculating the numbers of viable colony forming units (cfu) and generation time was calculated from the logarithmic exponential phase of the growth curve [12].

Reaction of rhizobial isolates were tested on media supplemented with Congo red at concentration (25 μg/ml). Acid or alkali production test was performed on YEMA-BTB medium (0.5%) bromothymol blue (BTB) indicator [13]. The ability of isolates to produce acid or alkaline in this media were evaluated by observed the color change around growing colonies; fast growing rhizobial strains produce acid in this medium, turning the medium yellow and slow growing Rhizobia produce alkali, which turns the medium blue. The isolates were also tested for growth on peptone glucose medium [14].

Tolerance of the isolates to salinity was tested on YEAM medium supplemented with, 1, 2 and 3 % (w/v) NaCl and that of acidity/ alkalinity on the same medium adjusted to pH of 4.5, 5 and 7.5. They were also grown on the same medium incubated at 25, 30 and 35°C to evaluate their tolerance to heat [15]. The intrinsic antibiotic resistance (IAR) of the strains was performed according [16]. The antibiotics were prepared at different concentrations (μg/ml); Ampicillin (10), penicillin (10), kanamycin (15) and Rifampicine (10). Resistance to heavy metal was tested on YEMA medium containing; K₂Cr₂O₇ 50, MnCl₂ 50, AlK(SO₄)₃ 25 and MnSO₄ 75 (μg/ml) of water by Maatallah J, et al. [16].

The ability of strains to utilize different carbohydrates (1% (w⁄v) as the sole carbon source was tested on basal media containing (g/l); K₂HPO₄ (1), KH₂PO₄ (1), FeCl₃.6H₂ (0.01), MgSO₄.7H₂O (0.2), CaCl₂ (0.1; (NH₄)₂SO₄ (1) and agar (15). Carbon sources such as galactose, maltose, lactose and raffinose were used. Likewise, methionine, tyrosine, thiamine and riboflavin as nitrogen substrates were tested on the same basal medium after replacing ammonium sulfate (1g/l) and reducing mannitol to a final concentration of 0.5 % (w/v) according to Amarger N, et al. [17].

Results and Discussion

Morphology and related growth characteristics

The selected tested isolates exhibited colony diameter ranges from 1.5- 4.1 mm and the largest diameter development at isolate 29P4S2-a and 87P1S1were more pronounced (Table 1). The isolates illustrate gram negative nature which ensures that isolates free from gram positive bacteria. The isolates also formed colony with circular shape, entire margin, mobile motility, white large creamy mucoid to watery small creamy mucoid texture with different level of mucus production. Which conform the typical feature of Mesorhizobium isolates characteristics (Table 2) (Figure 1).

Table1: List of isolates and regions of culture collection.

Table2: General colony growth characteristic of 15 rhizobia isolates from major chickpea growing regions of Ethiopia.

Figure 1: (a) Size, Shape, appearance and gram reaction and (b) biochemical characteristics.

All isolates changed the color of YEMA supplemented with BTB to yellow indicating that they are acid producers [18]. The CR absorption test also indicated that none of the isolates absorbed CR in YEMA plates; this is distinctive character of rhizobia with only few exceptions [11]. On the other hand, none of the tested isolates manage to grow on PGA plates.

The isolates revealed doubling time ranging from 1.5 h to 3.4 h. The isolate 13P3S2 and ET1were typically grown faster with mean generation time (1.2, 1.6h) respectively, whereas 76P3S1 were found to be moderately fast grower with mean generation time (1.6 h). Isolate 20P2S1 and ET13 had intermidiate growth rate.

All isolates revealed none absorbance of Congo red and did not grow on Peptone glucose agar with Bromocresol Purple, that was an indicative of rhizobia characteristics. Most of the isolates changed the colour of YMA medium supplemented with BTB to yellow and four isolates displayed blue that predicts the ability of the strains in acid or alkali production.

Most of the different isolates showed similar growth pattern to previous study in chickpea rhizobia. Thus, the report of Romdhane SB, et al. [19] described that all of chickpea nodulating rhizobia isolates from Tunisia showed the same colony morphology and growth rate on YMA medium. Whereas on other study Mesorhizobium ciceri [3] were found specific chickpea symbionts distinguished as slow growing rhizobia. According to Sharma A, et al. [20] the isolates which produced yellow colour colonies would have been classified as acid producer/fast growers, whereas isolates produced blue colour colonies indicated alkali producer/slow growers.

Most isolates (87%) were able to grow at 1% NaCl concentration, whereas 73% and 60% of the isolates were tolerant to 1%,2% and 4% NaCl, respectively (Table 3). A previous study in Portugal showed that Chickpea isolates showed a better growth with 1.5% NaCl, but inhibited at 3% NaCl concentrations [21]. More isolates grew at pH 7.5 (93%) than at pH 5 (66%), but fewer strains were tolerant to pH 4.5 (33%) (Table 3). Other studies showed that chickpea rhizobia were tolerant to pH 5 to 9.5 [22]. About 40% of the isolates grew at optimum temperature up to 35°C, while 80% were tolerated 25°C. Chickpea Mesorhizobia tolerant to different temperature levels between 15°C, 37°C were isolated elsewhere [23].

Table 3: Differentiation of stress tolerance, carbon and nitrogen substrate utilization pattern of isolates.

Most isolates were resistant to high kanamycin (80%), Rifampicine (60%) and Ampicillin (11%) (Table 3). Majority of the isolates exhibited (66%) resistance to the heavy metal MnSO4, but fewer isolates were tolerant K₂Cr₂O₇ (53%), followed by MnCl₂ (46%). Muletta D and Assefa F [24] reported that most Ethiopian chickpea rhizobia isolate were resistant to common antibiotic and heavy metals. The pattern of utilization of carbon substrates showed that most isolates (60%) were able to grow using galactose as carbon sources; followed by a number of isolates (66%, 45%) utilizing maltose and lactose, respectively cellulose (data not shown). Similarly, the majority of isolates better utilized the amino acids methionine (60%) and tyrosine (53%). Other studies showed chickpea isolates displayed high utilization of carbon and nitrogen substrates [16,24].

 

Conclusion

The study showed the presence of a predominant chickpea Rhizobia accompanying tolerance to various stress conditions and nutritional versatility. Isolates such as; ET1 and ET3 displayed wide range of stress tolerance and versatile substrates utilization. In general, the tested isolates showed dominance stress tolerance, carbon and nitrogen assimilation features, inherent antibiotic and heavy metal resistance. This might provide a greater tolerance to adverse environments and compete against the possible influence of ineffective indigenous Rhizobia for nodulation and nitrogen fixation. Thus, the results of this study highlighted the potential of such isolates to further evaluate at green house and wide environment field trials for the selection of elite inoculant strains that could presumably enable to applied under different soil and environmental conditions.

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