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Research Article - (2021) Volume 12, Issue 11

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 Date: Sep 10, 2021 / Accepted Date: Nov 20, 2021 / Published Date: Nov 27, 2021

Abstract

Chickpea (Cicer arietinum L.) is a major legume crop in Ethiopia and provide multiple benefits, due to high nutritive value as well as the ability of the crop to enrich nitrogen poor soils due to biological nitrogen fixation with different Rhizobia isolates. However, the effectiveness of the isolates varies due to inherent feature, stress tolerance and substrate utilization characteristics of the isolates. This necessitates the screening of the basic properties of the isolates under in vitro laboratory conditions. To this effect, 15 indigenous isolates from chickpea growing regions were tested for in vitro basic features, stress tolerance and substrate utilization properties. There were variations in morphological features, stress tolerance and nutritional diversity among isolates. The isolates also formed colony with circular shape, entire margin, white large creamy mucoid to watery small creamy mucoid texture. Chickpea isolates showed broad range (0-100%) salt tolerance to different NaCl concentrations. The isolates grown in moderately acidic pH 4.5 to alkaline pH 7.5 ranging from (25-50%). Subsequently the isolates were grown at optimum temperature up to 37°C level range from (25-100%). The isolates were more tolerant to the tested antibiotics (0-75%) and resistance to the heavy metal (0-100%). In addition, chickpea isolates better utilized the carbohydrates (0-100%) and similarly, the amino acids (25-75%). All taken together, the data provided an important complement to select representative isolates competitive in the soil which is a one of the desirable characteristics for inoculant isolates selection for effective nitrogen fixation.

Keywords

Chickpea; Characteristics; Isolate; Morphology; Rhizobia

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).

No Isolates Region Latitude Longitude Elevation Soil pH
1 ET1 Amhara, South Gonder 11°27'58.3"N 38°12'46.6"E 2795 6.9
2 ET3 Oromia, East Shewa 8°49'31.7"N 38°59'25.4"E 1943 6.7
3 56P2S1 Amhara, South Gonder 11°55'9.3"N 37°52'36.9"E 1995 5.4
4 29P4S2-a Amhara, East Gojam 10°42'47.3" 38°10'30.6"E 2541 6.4
5 20P2S1 Oromia, West Shewa 8°39'21.1"N 38°29'2.8"E 2207 7.3
6 ET13 Amhara, North Gondar 12°11'15.4"N 37°40'24.8"E 1889 7.0
7 58P2S1 Amhara, South Gonder 11°29'16.6"N 38°12'45.8"E 2870 6.2
8 13P3S2 Oromia, West Shewa 8°46'10.9"N 38°39'5.6"E 2231 7.5
9 76P3S1 Amhara, North Gondar 12°20'59.4"N 37°11'57.9"E 1808 7.8
10 68P1S1 Amhara, North Gondar 12°26'13.2"N 37°30'25.3"E 1930 6.8
11 87P1S1 Oromia, West Shewa 8°43'6.1"N 38°15'53.5"E 2127 6.3
12 ET7 Amhara, North Gondar 12°28'35.9"N 37°22'57.5"E 1988 7.4
13 ET19 Amhara, South Gonder 12°5'20.4"N 37°45'17.9"E 1865 6.4
14 42P3S1-a Amhara, North Gondar 12°28'35.9"N 37°22'57.5"E 1988 5.8
15 ET14 Amhara, North Gondar 12°11'14.7"N 37°40'24.8"E 1888 6.4

Table1: List of isolates and regions of culture collection.

No Isolates Colony
size (mm)
Colony
appearance
Shape BTB reaction MGT
(hrs)
Peptone glucose
1 ET1 2.4 Large creamy mucoid Domed Yellow 1.5 NG
2 ET3 2.7 Large creamy mucoid Domed Yellow 2.0 NG
3 56P2S1 2.2 Small creamy mucoid Domed Blue 2.5 NG
4 29P4S2-a 4.1 large creamy mucoid Domed Yellow 3.4 NG
5 20P2S1 1.5 Small creamy mucoid Flat Yellow 1.7 NG
6 ET13 2.0 Waterless creamy mucoid Domed Yellow 1.7 NG
7 58P2S1 2.1 Small creamy mucoid Conical Yellow 3.2 NG
8 13P3S2 1.5 Small creamy mucoid Domed Blue 1.2 NG
9 76P3S1 1.5 Milky large mucoid Domed Blue 1.6 NG
10 68P1S1 1.6 Large creamy mucoid Flat Yellow 3.3 NG
11 87P1S1 3.5 Large creamy mucoid Conical Yellow 2.1 NG
12 ET7 2.0 Milky less mucoid Domed Yellow 3.4 NG
13 ET19 2.8 Large creamy mucoid Flat Blue 1.9 NG
14 42P3S1-a 1.8 Small creamy mucoid Domed Yellow 2.8 NG
15 ET14 1.6 Small creamy mucoid Domed Yellow 2.2 NG

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

plant-pathology-microbiology-biochemical

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].

Characteristics ET1 ET3 56P2S1 29P4S2-a 20P2S1 ET13 58P2S1 13P3S2 76P3S1 68P1S1 87P1S1 ET7 ET19 42P3S1-a ET14 Total (%)
Salt Tolerance                                
1% + + + + + + + + + + + + + - - 87
2% + + - - + - - + + + + + + + + 73
3% + + - - + + + - - - - + + + + 60
pH Tolerance                                
pH 4.5 - - - + - + - + - - + - + - - 33
pH 5.0 + + + - + - - - + + + + - + + 66
pH 7.5 + + + + + + + + + + - + + + + 93
Temperature                                
25°C + + + + + + + + - - + + + + - 80
30°C + - + - + - - + + + + + + + + 73
35°C - + + - + - +   - + - - - - + 40
Antibiotics                                
kanamycin + + + + - + + + + - + + + + - 80
Rifampicine - - + - + - + + + - + + + + - 60
Penicillin + + - + + + - - - + - - - - - 40
Ampicillin - - - - - - + - - + - + - - - 11
Heavy Metals                                
MnSO4 + + - + + + - + - + + + - - + 66
K2Cr2O7 + - - + + + + + - + - - - + - 53
MnCl2 + - - - - - + - - - + + + + + 46
AlK(SO4)3 + + - - - + - - - - + - - - - 26
Carbohydrate 100 75 50 0 75 100 75 75 0 75 75 75 50 50 25  
Amino acid 50 50 50 25 25 25 25 75 25 50 50 50 50 50 50  

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.

References

Citation: Damtew ZM (2021) Morphological Variation and Basic Characteristics of Selected Indigenous Rhizobia Isolated from Major Chickpea (Cicer arietinum L.) Growing in Regions of Ethiopia. J Plant Pathol Microbiol 12:585.

Copyright: © 2021 Damtew ZM. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.