Journal of Plant Pathology & Microbiology

Ecological Competence, Plant Growth Promoting and Symbiotic Characteristics of Different Mesorhizobium Strains Nodulating Chickpea (Cicer arietinum L.) from Ethiopia

Zehara Mohammed Damtew^1*, Asnake Fikre², Douglas R Cook³, Alex Greenlon⁴, Edward Marques⁵, Eric J Wettbergg⁶, Kassahun Tesfaye¹, Noelia Carrasquilla Garcia³ and Fassil Assefa^{1 1}College of Natural Sciences, Addis Ababa University, Addis Ababa, Ethiopia
²Debre Zeit Agricultural Research Center, Ethiopian Institute for Agricultural Research, Ethiopia
³Department of Plant Pathology, University of California, United States
⁴Environmental Science, Policy and Management, University of California, USA
⁵Department of Biological Sciences, Florida International University, Florida, USA
⁶Department of Plant and Soil Science, University of Vermont, Vermont, USA
^*Correspondence: Zehara Mohammed Damtew, Ethiopian Institute of Agricultural Research, Debre Zeit Agricultural Research Center, Debre Zeit, Ethiopia, Tel: +251911047518, Email:

Received: 08-Jun-2020 Published: 12-Aug-2020, DOI: 10.35248/2157-7471.20.11.508

Introduction

Nitrogen and phosphorus are most commonly limiting nutrient in agricultural crop production in in sub-Saharan Africa including Ethiopia. Chickpea (Cicer arietinum L.) like most legume plants access reduced nitrogen from the soil, through their roots, in the forms of nitrate and ammonium [1]. Chickpea is one of the popular pulse crops used for crop rotation for it fixes nitrogen in association with root nodule bacteria from the genus Mesorhizobium [2]. Chickpea requires about 13 to 41 kg /ha inputs of nitrogen for growth and development from which it derives 70% of its N through symbiotic N₂ fixation [3,4]. Apart from biological nitrogen fixation, root nodule bacteria are one of the important plant growths promoting rhizobacteria (PGPR) such as Pseudomonas and Bacillus that enhance plant health, productivity and provide addition of fertilizers [5,6]. Indirect act as biocontrol against phytopathogens through various forms of antagonism like competition, production of antibiotics, lytic enzymes and hydrogen cyanide [7]. Thus, it is essential to understand existence of native rhizobia in the soils, genetic variation in bacterial strains and symbiotic response of the cultivar enables to distinguish their symbiotic efficiency [8]. In Ethiopia [9] isolated 39 root nodule bacteria from chickpea growing areas of the eastern, southeastern and southern parts of the country, of which 23 isolates (59%) were identified as chickpea root nodule bacteria with the same identification methods. However, effective Mesorhizobium strains compatible to multiple varieties of the crop inoculation have not been intensively characterized from representative regions of Ethiopia and we lack an understanding of diverse Mesorhizobium strain in relation to their ecophysiological competence. Therefore, the purpose of this study is identifying competitive Mesorhizobium strains which displayed large spectrum persistence adaptive mechanism with different types of chickpea varieties to addressed developing inoculum towards promising impact on chickpea production.

Materials and Methods

Source of strains and growth conditions

Twenty strains were selected from the phylogeny of whole genome sequenced 64 strains belonging to six Mesorhizobium spp. which were isolated from root nodules collected from major chickpea growing regions of Ethiopia and compiled with global level population genomics [10]. The strains have been deposited in culture collections at Plant Pathology Laboratory of Davis, University of California, USA and Addis Ababa University. NCIB assembly accession links presented in (Dataset S1) accompanying studies by Greenlon et al. [10] (Table 1).

Table 1: List of Mesorhizobium strains and origin of culture collection for strains used in Phenotypic and symbiotic characterization.

Eco-physiological characteristics

Tolerance of the Mesorhizobia strains to salinity were tested on YAM medium supplemented with, 2, 3 and 4% (w/v) NaCl and tolerance to acidity/alkalinity on the same medium adjusted at pH of 4%, 5% and 10% using 1N HCl and NaOH before autoclaving. They were also grown on the same medium and incubated at 35°C, 37°C and 40°C to evaluate their tolerance to heat stress [11]. The intrinsic antibiotic resistance (IAR) of Mesorhizobium strains was performed according [12]. Various filter sterilized antibiotics were prepared at different concentrations (μg/ml); Chloramphenicol (10), Streptomycin (50), Nalidixic acid (10), Erythromycin (10), Neomycin (10) and tetracycline (10). Resistance to heavy metal was tested on YEMA medium containg; CoCl₂ 25, CuCl₂ 50, ZnCl₂ 50, AlCl₃ 250, Pb (CH₃COOH)₂ 250 and NiSO₄ 100 (μg/ml) of water. Resistance was recorded when visible growth was detected on the medium.

Carbon and nitrogen substrates utilization

The ability of strains to utilize different carbohydrates (1% (w⁄ v) as the sole carbon source was tested on basal media contains (g/l); K₃HPO₄ (1), KH₂PO₄ (1), FeCl₃.6H₂ (0.01), MgSO₄.7H₂O (0.2), CaCl₂ (0.1; (NH₄)₂SO₄ (1) and agar (15). Carbon sources included Sucrose, α-cellulose, Trehalose, DGalactose, D-Xylose and D-Sorbitol. Likewise, the ability of the strains to utilize nitrogen substrates such as; L-lysine, LPhenylanine, L- Tryptophan, L- Leucine, L- Argenine and Glycin was tested on the same basal medium after replacing ammonium sulfate (1 g/l) and reducing mannitol to a final concentration of 0.5% (w/v) according to Amarger et al. [13].

Plant growth promoting properties of strains

Indole acetic acid (IAA) production: Strains were allowed to grow on YMA broth supplemented with filter sterilized Ltryptophan (2 g/l) and grown on orbital shaker at 200 rpm at room temperature for 4 days to test their ability to produce IAA [14]. The cultures were centrifuged at 10000 rpm for 15 min from which 2 ml of the supernatant was mixed with 100 μl of 10 mM orthophosphoric acid supplemented with Salkowaski reagent (1 m of 0.5 M FeCl₃ in 50 ml of 35% HClO₄) in a ratio of 2:1. Samples turned pink color were considered positive indicator for IAA and the absorbance was measured using spectrophotometer at 530 nm and the amount of IAA produced was calculated by comparing with standard curve constructed from different concentrations of IAA.

Production of hydrogen cyanide (HCN): Strains were inoculated in YMA Plates supplemented with 4.4 g/l of glycine to test their ability produce hydrogen cyanide [15]. A Whatman filter paper no.1 moisturized in picric acid solution (2.5 of picric acid and 12.5 of Na₂CO₃ (g/l) of water dissolved reagents) was placed in the upper lids of the Petri plate. The plates were sealed with parafilm and incubated at 28°C. Change in colour from yellow to light brown was considered as weak, brown as moderate and reddish brown as strong was recorded to show the levels of hydrogen cyanide production.

Phosphate solubilization on solid medium: Qualitative estimation of phosphate solubilization was performed using three inorganic phosphate sources, tricalcium phosphate Ca₃(PO₄)₂, Aluminum phosphate (AlPO₄) and Iron phosphate (FePO₄) plate assay. Active culture suspension of 10 μl (~108 cells/ml) of each strain was spot inoculated on Pikovskaya’s agar medium to assess tricalcium phosphate solubilization potential. The medium consisting of ingredients in g/l; glucose (10), Ca₃(PO₄)₂ (5), (NH₄)₂SO₄ (0.5), MgSO₄.7H₂O (0.1), yeast extract (0.5), NaCl (0.2), MnSO₄.2H₂O (0.002), FeSO₄.7H₂O (0.002) and agar (15) according to Pikovskaya. Simultaneously, the strains were inoculated National Botanical Research Institute ’ s Phosphate medium to assess their solubilization ability of Aluminum phosphate and iron phosphate. The medium contained the following ingredients in g/l: glucose (10), AlPO₄ or FePO₄) (5), MgCl₂.6H₂O (5), (NH₄)₂SO₄ (0.1), KCl (0.2), MgSO₄.7H₂O (0.25) and agar (15) according to Perez et al., [16]. The formation of clear halo zone around colonies and the solubilization index (SI)=(colony + halo zone) to the colony diameter in mm was recorded.

Phosphate solubilization on liquid medium: Based on their solubilization index, strains were selected to perform quantitative estimation of phosphate solubilization in PKV (Ca₃(PO₄)₂) and NBRIP (AlPO₄ and FePO₄) broth. The assay was carried out by inoculating active culture suspension of 100 μl (~108 cells/ml) of the respective strains using 100 ml of the respective broth in 250 ml Erlenmeyer flasks. Then the flasks were incubated on a rotary shaker at 200 rpm at room temperature for 8 days. Five ml of the supernatant was taken on the 4th, 8th day to measure pH with pH meter the amount of phosphate released using phosphomolybdate method [17] standard method. For the latter, the supernatant was centrifuged at 14,000 rpm for 15 minutes and the amount of phosphate in the clear culture supernatant as well in control (without inoculation) was measured using spectrophotometer (540 nm). The amount of solubilized P (μg/ml) was quantified against a standard curve constructed from known concentratins of Potassium dihydrogen phosphate (KH₂PO₄).

Symbiotic effectiveness screening at greenhouse

The experiment was conducted in pot sand culture in the greenhouse at Debre Zeit Center, Ethiopian Institute of Agricultural Research (EIAR). Natoli and Arerti Chickpea seeds were treated with 70% ethanol (30 sec), surface sterilized in 2% sodium hypochlorite (3 min) and rinsed five times with sterile water. Seeds were germinated on 1% (w/v) water agar at 28°C and transplanted into surface sterilized 3-kg capacity pots filled with acid washed and sterilized white sand [18]. Each seedling was inoculated with 1 ml liquid inoculum (~109 cells/ml) of each test Mesorhizobium strain and 2 reference strains (Ethiopian commercial strain EAL 029, Tunisia strain Ha. Ata). The experiment was done in Complete Randomized design with three replications. The seedlings were provided weekly with Nfree nutrient solution and the N-fertilized pot at a rate of 70 μg N ml-1 KNO₃ solution once a week [19].

Data scoring and plant sample analysis

Plants were uprooted after 45 days of planting to record number of nodules, nodule dry weight and shoot dry weight. Relative effectiveness of strains was calculated using the formula, RE=(inoculated plant shoots dry weight/shoot dry weight of nitrogen supplemented plant) × 100 [20]. Nitrogen fixing effectiveness was classified as highly effective >80%; effective 50 to 80%; low effective 35 to 50% and ineffective < 35%.

Data analysis

Values were given as means for triplicate samples. Analysis of variance (ANOVA) for comparison between the treatments for shoot dry weight, nodule number and nodule dry weight was performed using the statistical software SAS version 9.3. The difference among treatment means was compared by high range statistical domain (HSD) using Tukey test at 5% probability level.

Results and Discussion

Physiological and biochemical characteristics

Almost all strains (91%) were able to grow at 1% NaCl concentration, whereas 50% and 35% of the strains were tolerant to 3% and 4% NaCl, respectively (Table 2). Among the groups, M. genospecies 7A strain 27P3S2 affinity to M. ciceri, M. genospecies 1B strain 45P4S1 closest relative to M. loti, M. genospecies 2A strain 43P2S1 and M. genospecies 2A strain 46P3S2 affinity to M. plurifarium showed broad range salt tolerance to different NaCl concentrations. This might be due to induced overproduction of low molecular weight proteins which help the cells to osmotic adjustment to intracellular water. Chickpea isolates showed a better growth with 1.5% NaCl [21], whereas the bacterial growth severely affected at 3% NaCl concentrations. Previous studies in Ethiopia [22] showed 11% of the tested chickpea isolates tolerated 5% NaCl.

Table 2: Eco-physiological characteristics of chickpea nodulating Mesorhizobium strains.

Many strains were grown more at pH 10 (85%) than pH 5 (60%), but fewer strains were tolerant to pH 4 (25%) (Table 2). Five strains from M. genospecies 1B strain 45P4S1, M. genospecies 2A, M. genospecies 4A strain ET26, strain 43P2S1, M. genospecies 4A strain 22P5S2 and M. genospecies 1E strain 38P4S2 affinity to M. loti and were tolerant to pH 4. Previous studies in Ethiopia showed that chickpea rhizobial isolates grew well in moderately acidic pH5 to alkaline pH 9; while sensitive acidity pH 4 [22].

The strains showed growth at optimum temperature 35 up to 37°C, but fewer strains (20%) were tolerant to 40°C (Table 2). M. genospecies 1B strain 45P4S1, M. genospecies 4A strain ET26 and M. genospecies 4B strain 80P4S2 resemble to M. amorphae and M. genospecies 2Astrain 43P2S1showed rigorous growth on extreme temperature (40°C). Concurrently, Tassew Sirage et al. reported that 50% of the chickpea rhizobia strains were tolerant to 40°C isolated from Ethiopia [23].

The majority of the Mesorhizobium strains (60-90%) were resistant to lower concentrations of antibiotics, except tetracycline. Most strains exhibited high resistance to high concentrations of erythromycin (60%), nalidixic acid (65%) and streptomycin (50%) (Table 3). M. genospecies 8A strain ET1, M. genospecies 9A strain 2P3S1-b and M. plurifarium affinity strains group were more tolerant to high concentrations of the tested antibiotics (33-44%) than M. amorphae affiliated strains (11-33%). Several studies showed chickpea isolates were resistant to nalidixic acid and erythromycin [24,25].

Table 3: Nutritional versatility, intrinsic antibiotic resistance and heavy metals pattern of different Mesorhizobium species group.

Most of the Mesorhizobium strains exhibited (60%) resistance to the heavy metal CoCl₂, but fewer isolates were tolerant CuCl₂ (45%), followed by ZnCl₂ (30%) and highly sensitive to Al (10%) and Pb (5%). M. genospecies 9A strain 2P3S1-b, M. genospecies 7A strain 27P3S2 strains were highly resistant to most heavy metals (Table 3). The resistance of chickpea rhizobial isolates to Co and Cu was previously reported [24,26].

The pattern of utilization of carbon substrates showed that most strains (60%) were able to grow using D-Sorbitol and D-Glucose as carbon sources; followed by a number of strains (50%, 45%) utilizing Sucrose and Trehalose respectively and none of the strains did not utilize α-cellulose (Table 3). Earlier study indicated that chickpea rhizobia strains were more known in utilizing the carbohydrates [24,27].

Similarly, the Mesorhizobium strains better utilized the amino acids Phenylanine (60%), Leucine (40%) and Argenine (35%). Few strains such as M. genospecies 9A strain 2P3S1-b resemble to M. sp. LSJC280B00, M. genospecies 4B strain 80P4S2 and M. genospecies 2A strain 43P2S1 utilized lysine. Relative strains to M. plurifarium, M. sp. LSJC280B00 and M. australicum utilized Glycine. Earlier study on chickpea isolates from Ethiopian soils showed no growth of isolates on glycine were reported [22], whereas Kucuk et al. observed that few isolates utilized glycine [26].

Plant growth promoting properties of Mesorhizobium strains

The twenty different Mesorhizobium groups were tested for their ability to solubilize various inorganic phosphate sources, produce indole acetic acid and HCN (Table 4). With regard to IAA production, all strains produced the phytohormone ranging from 7.7 (μg/ml) to 28.4 (μg/ml) (Table 4). The M. genospecies 8A strain ET4 relative to M. australicum showed the highest score of IAA (28.4 μg/ml), indicating a 4-fold difference between the highest IAA and the lowest producer (7.7 μg/ml) M. genospecies 3A strain 10P4S2. Other studies showed M. ciceri and M. loti chickpea isolates displayed high IAA production (47.38, 45.2 μg/ml) by Brigido et al., [21]. Previous chickpea isolate from Ethiopia showed (12.3 and 58.0 μg/ml) IAA production respectively [22,27]. Three strains (15% of the isolates); M. genospecies 3A strain 80P4S2, M. genospecies 4A strain 22P5S2 and M. genospecies 1B strain 45P4S1 solubilized tricalcium and aluminium phosphates with solubilization indices ranging from 0.34-1.17. No strain solubilized iron phosphate and produced HCN.

Table 4: Plant growth promoting properties of chickpea nodulating Mesorhizobium strains.

Although the solubilization indices (SI) were comparable (1.12-1.3), the population density of phosphate solubilizing Mesorhizobium strains (15%) was much lower than the number (44%) reported by Mulisa Jida et al. and 30% enumerated by Daniel et al. [28]. The SI of isolates in Ethiopia were slightly lower than recorded from SI (1.42) displayed by Mesorhizobium ciceri isolated from Iran [29]. The ability to solubilize inorganic phosphate suggesting the ability to solubilize inorganic phosphate may be species related and may constitute an adaptive mechanism against the phosphorous deficiency in acidic and alkaline soils [21].

The amount of phosphorus released by these strains in a liquid culture (Table 5). The data showed, M. genospecies 4B strain 80P4S2 was able to release the highest amount of available soluble phosphates from Ca₃(PO₄)₂ (118.0 μg/ml) and FePO₄ (93.3 μg/ml) after 8 days of incubation. The phosphates released from tricalcium after 8 days of incubation for three strains ranged from (29.0 to 118.0 μg/ml) and at aluminium phosphate (41.67 - 93.3 μg/ml). The M. genospecies 4B strain 80P4S2 was found the better strains in solubilization of both inorganic phosphate sources as it released phosphate constantly until 8 days of incubation.

Table 5: Tricalcium and Aluminium phosphate solubilization efficiency of Mesorhizobium strains.

The data revealed a slight decrease in pH as incubation time increased and the maximum pH was observed 6 and the least 4.3. The two strains exhibiting the highest levels of Ca₃(PO₄)₂ and AlPO₄ solubilization in liquid medium were also shown to produce large solubilization halos when tested in solid medium. The amount of phosphate released in this study was moderate compared to release from tricalcium was ranged (70 to 295 μg/ml) after 8 days of incubation [27].

Nodulation and symbiotic effectiveness of strains under greenhouse conditions

The inoculants showed significant variation in nodulating the two varieties ranging from (31-62) number of nodules per plant (Table 6). However, more nodules were recorded from the Natoli variety (32-62 nodules) compared to fewer nodules (31-46) formed on Arerti variety (Table 6). This might indicate that the varieties responded differently to individual strains. The strains induced nodule dry weight within the range of 55 mg/plant to 557 mg/plant on two varieties. Earlier study on chickpea isolates from Ethiopian soils showed nodule dry weight variation between 44 mg/plant to 497 mg/plant [30]. Study at Sudan showed that 35% nodules dry weight increased rate over the uninoculated control [31].

Table 6: Inoculation response on different nodulation traits of chickpea varieties at greenhouse.

The Natoli variety produced shoot dry weight in the range of (0.62-1.38 g) per plant on both varieties. More shoot dry matter accumulation was recorded from the Natoli variety (0.78-138 g) compared to fewer shoot dry matter (0.62-1.18 g) formed on Arerti variety. The shoot dry matter in this study did not exceed the values reported (0.6-1.36 g/plant) for chickpea isolates by Mulisa Jida and Fasil Assefa [22]. Symbiotic effectiveness in relation to shoot dry matter by the inoculated plants in reference to the nitrogen fertilized control, 85% of the strains were highly effective with shoot dry matter accumulation of >80% on both varieties. Equivalently the remaining (30%) were categorized as effective with shoot dry matter accumulation of 50-80% on Natoli and Arerti varieties. Study at Ethiopia by Wubayehu Gebremedhin et al., [9] showed 125% symbiotic effectiveness and recently Tassew Siraj et al., [23] reported (70%) on Natoli and 21% Arerti symbiotically highly effective and effective isolates. Significant variations were not observed for shoot nitrogen content; M. genospecies 2A strain and M. genospecies 4A strain ET26 accumulated relative shoot nitrogen content (1.2%) on both respective varieties. Most strains accumulated low nitrogen content compared to shoot nitrogen concentrations in inoculated chickpea plants [27].

The overall eco-physiological, nutritional PGP and symbiotic tests (Table 7) showed that M. genospecies 9A strain 2P3S1-b closest affinity to M. sp. LSJC280B00 and M. genospecies 4B strain 80P4S2 relative to M. amorphae showed wide range of ecophysiological tolerance and versatile substrates utilization (Table 6). Subsequently, M. genospecies 1B strain 45P4S1 closest relative to M. loti, M. genospecies 2A strain 43P2S1 affinity to M. plurifarium, M. genospecies 4A strain ET26 relative to M. amorphae indicating pronounced competitiveness.

Table 7: Overall rating of eco-physiological, nutritional and PGP characteristics of chickpea nodulating Mesorhizobium strains.

Conclusion and Recommendations

The finding reveals that Ethiopian Mesorhizobium strains typically have high eco-physiological and nutritional variability that are vital to local adaptation. Three strains have multiple growth promotion properties; solubilization of phosphates from calcium and aluminium phosphates that have the capacity to mobilize phosphorus in the soil. All strains produced the growth development phytohormone IAA. It was interesting to note that more than 90% of the strains showed highly effective symbiosis on both Natoli and Arerti varieties interms of relative shoot dry matter accumulation by the inoculated plants in reference to the nitrogen fertilized control plants. The overall eco-physiological competence and symbiotic effectiveness suggests the potential of these strains to test in multi-location field trials for enhance nitrogen fixation activities in chickpea production.

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