E. brucei is a multipurpose tree legume which has been grown as a shade tree for coffee plantation and source of green manure in farmlands in the southern Ethiopia. In south Ethiopia, the plant has been mainly used as cover plant, green manure and mulching materials by small holder farmers and taken as low cost organic agricultural input. This is because E. brucei forms dual symbiotic association with rhizobia for nitrogen fixation and AMF for phosphorous nutrition [26, 42]. It has also been planted as biofence/land boundary fence and components of home gardens and forests in all parts of the country. Therefore, this work is initiated to enhance the growth, development and productivity of this multipurpose tree legume and to enrich its biomass nitrogen and phosphorous through application of phytobeneficial microorganisms to introduce improved and low cost agro-forestry practices.
This paper is a part of a long term plan of a work aimed to come up with improved agroforestry practices involving E. brucei. Improved agroforestry practices can be achieved through enhancement of growth, development and productivity, and enrichment of biomass with P and N status of E. brucei. In addition, sustainable and improved agroforestry practices could be achieved through application of low cost and eco-friendly phytobeneficial microbial inputs by exploring the rhizosphere of particular plant for phytobeneficial microorganisms namely Arbuscular mycorrhizal fungi, nitrogen fixing bacteria, phosphate solubilizing bacteria and microbes with multiple plant growth promoting traits.
In the present study, we recorded 39.14% inorganic phosphate solubilizing bacteria. All these bacteria exhibited clearly visible halo zones on PA plates containing TCP. The existence of soil microbes endowed with inorganic phosphate solubilizing capabilities in alkaline and/or acidic soils very important asset. These phytobeneficial microbes can solve P nutrient deficit by solubilizing and releasing P locked in Al-P, Fe-P and Mn-P in acidic soils and Ca-P in alkaline soil. Similarly, Amsalu et al [8] evaluated nine E. brucei root nodule bacterial endophytes for inorganic phosphate solubilization and reported six strains which exhibited TCP solubilization ability in PA medium through formation of clear halos. This shows that the root nodules of this particular host plant harbor a large number of phosphate solubilizing bacteria. The 43% of Ethiopia soils are acidic [43], hence in these acidic soils the inorganic phosphates are found adsorbed into sparingly soluble precipitates of FePO4 and AlPO4 [44]. Therefore, it is imperative to consider acidic soils of Ethiopia, while isolating phosphate solubilizing microbes as part of tropical and subtropical soils. Due to this fact, we included two different synthetic inorganic phosphate compounds (Al-P and Fe-P) in the preliminary screening representing sparingly soluble inorganic P sources which commonly found in soils as sole P sources.
In the present study, all the 119 bacterial isolates showed growth on PA medium supplemented with AlPO4 or FePO4, however, none of them formed visible clear halo zones around colonies. Dinic et al.[45] have reported corroborating findings among root nodule endophytic bacteria from French bean. These scholars have reported French bean root nodule bacteria that grew well on plates containing Al-P or Fe-P but did not form visible clear halos around colonies. The growth of bacterial colonies without exhibiting visible halos on plates containing Al-P or Fe-P might be attributed to the solubilization of very small amount of P which could be consumed by the bacterial isolates for their immediate growth[46, 47].
In this study, 7.6% of phosphate solubilizing bacterial isolates formed PSI values ≥4.0, 56.3%, formed PSI values 4>PSI>2.0,and 36.1% formed PSI values less than 2.0. According to Marra et al.[48], 7.6%, 56.3% and 36.1% of our isolates are high, intermediate and low phosphate solubilizers,respectively. Likewise, Muleta et al. [4] have reported phosphate solubilizing bacteria from natural coffee forest soil in southern western Ethiopia exhibiting comparable phosphate solubilization indices between 2.05 and 5.85 which corroborates to PSI values between 0.5 and 6.0 recorded by our isolates. This preliminary observation revealed the presence of potential inorganic phosphate solubilizing bacterial population in the root nodules of E. brucei which can be applied as low cost microbial inputs to enhance the growth, development and productivity of this particular host plant and enrich its biomass with P.
All the root nodules regardless of the geographic location harbored phosphate solubilizing bacteria in this study. The population concentration of phosphate solubilizing bacteria may be varied in different rhizospheres due to abiotic factors of the soil. Variations in the distribution of these bacteria with soil, climate and cropping history has been well documented[49, 50]. Similarly, Kim et al.[51] have reported effects of different soil properties like physical and chemical properties, organic matter, and P content and cultural activities on the population distribution of these bacteria in the soil.
With regard to diversity, the selected phosphate solubilizing bacterial isolates were clustered in to four genera namely: Achromobacter, Acinetobacter, Bacillus and Gluconobacter. All these genera are the first reports from the root nodules and rhizosphere of E. brucei. The presence of phytobeneficial traits like inorganic phosphate solubilization and IAA production is previously demonstrated for Acinetobacter [52], Achromobacter [53, 54]), Bacillus thuringiensis[54, 55]), and Gluconobacter [56].
All the evaluated isolates exhibited inherent inorganic phosphate solubilization potential by releasing variable amount of solubilized phosphate into NBRIP medium. Strains AU4 and RG6 (Acinetobacter soli) solubilized the highest, 120.36 and 112.82 mg L−1 from TCP at sampling day 6. Our isolates solubilized more than four times higher tricalcium phosphate compared to the control at sampling days 3 and 6 (Table 3). The phosphate solubilization in liquid media supplemented with TCP was concomitant to medium pH drops and phosphate solubilization seems to be due to the medium acidification which could be associated to either proton extrusion or organic acid secretion by the phosphate solubilizing bacterial isolates[57]. Several scholars have reported TCP solubilization by rhizobacteria with similar solubilization mechanism [4, 45, 58].
With regard to the quantity of solubilized phosphate released into NBRIP medium ,Chung et al. [39] have reported comparable quantity to our findings. However, scholars like Chen et al., [3] and Farhat et al.[59] have reported TCP solubilization by rhizobacteria yielding a greater amounts of solubilized phosphate compared to our findings. On the other hand, several scholars have reported rhizobacterial isolates solubilizing smaller amount of TCP in NBRIP medium compared to ours [9, 57].
We did not observe direct correlation between phosphate solubilization indices on the PA plates and the amounts of quantified solubilized phosphate in liquid medium supplemented with TCP. This may implies that the PSI values formed on agar plates do not necessarily guarantee phosphate solubilization efficiency in liquid medium.
Even though these phosphate solubilizing bacterial strains did not exhibit clear and visible halos on AlPO4 or FePO4 on solid medium, it has been previously demonstrated that many isolates which did not produce visible halos on agar plates solubilized different types of insoluble phosphates in liquid media [60]. Therefore, we evaluated the inorganic phosphate solubilizing ability of the selected isolates in AlPO4 or FePO4 supplemented NBRIP liquid medium.
In line to Gupta et al [60], all our selected isolates have shown mobilization of P from these insoluble inorganic phosphates in liquid media. Strains RG6 and AU4 (A. soli) solubilized the highest quantity of Al-P, 102.14 and 99.66 mg L−1, respectively at sampling day 6. Our isolates increased AlPO4 solubilization between three and twelve times higher compared to the controls at sampling day 3, and it was between six and fourteen times higher at sampling day 6 (Table 3). We recorded a higher inherent AlPO4 solubilization potential by our isolates compared to Chung et al.[39] who have reported isolates that solubilized P between 3.7 and 13.8 mg L−1. Likewise, Son et al.[61] have identified bacterial isolates from soya bean rhizosphere in Korea which solubilized small amount of phosphate (19 mg L−1) from AlPO4 in five days incubation. We also recorded drops in the medium pH across sampling periods which are concomitant with increased phosphate solubilization. Several scholars have reported similar medium pH dropping patterns in AlPO4 supplemented medium which is comparable to our results [4, 45, 57].
In the case of FePO4 solubilization, the highest amount of solubilized P, 96.07 mg L−1 was released by isolate RG6 (A. soli) followed by EN5 (G. cerinus) which solubilized 95.14 mg L−1 P. Our isolates exhibited between nine and nineteen times higher Fe-P solubilization at sampling day 3 and between eleven and twenty two times higher at sampling day 6 (Table 3) compared to the controls. The similar medium pH dropping trends were also recorded in FePO4 supplemented NBRIP medium like other inorganic phosphate sole sources used in this study.
We observed significant but inverse (r= -0.66, p<0.05) correlation between medium pH drops and incubation period. However, unlike the other two phosphate sources, we did not observe significant correlation between pH drops and the amount of solubilized phosphate in the case of FePO4 solubilization apart from medium acidification. This may indicate the presence of alternative and different inorganic phosphate solubilization mechanisms by rhizobacteria. Similarly, Son et al. [62] have evaluated FePO4 solubilization by rhizobacteria in NBRIP medium and did not find relationship between phosphate solubilization and drops in pH values which is in agreement with our findings. Similarly, Baliah et al.[50] have also observed drops in the pH of NBRIP medium due to bacteria inoculation, however, the pH reduction did not exhibit consistent correlation to the amount of solubilized phosphate. NBRIP medium acidification and pH drops associated with FePO4 solubilization are well documented by several scholars [4, 45, 57, 58]. Muleta et al. [4] have suggested that the medium acidification could be due to synthesis of diverse organic acids by the bacterial isolates by consuming the original carbon sources in the medium.
In order to achieve our long term goals, the application of potential microbial inputs to improve E. brucei based agroforestry practices is crucial. In this context, in the present work, we identified and characterized and evaluated E. brucei root nodule bacteria for multiple plant growth promoting traits in addition to inorganic phosphate solubilization.
About 40.3% of our phosphate solubilizing isolates produced IAA as confirmed through formation of pink coloration. However, the strength of the pink coloration in qualitative determination and the quantitified amount of IAA produced were varied among the bacterial isolates. The highest quantity of IAA, 0.313 mg L−1 was produced by isolate DM17 (Bacillus thuringiensis) followed by EN6 (Gluconobacter cerinus) which produced about 0.266 mg L−1. Amsalu et al. [8] have evaluated nine E. brucei root nodule endophytic bacteria and have reported 88% IAA producers. In addition, our isolates produced a greater amount of IAA (0.075 to 0.313 mg L−1) compared to Andrade et al.[62]who have reported phosphate solubilizing isolates producing IAA between 5e−5 mg mL−1 and 0.05 mg mL−1. Tariq et al. [9] have also showed root endophytes of pea plant synthesizing IAA between 8.6e−4 mg mL−1 and 0.016 mg mL−1.
IAA has been implicated in every aspect of plant growth, development and phytopathogen defense responses[63]. IAA synthesis by rhizobacteria increases plant root surface area and length, and consequently; increases the plant access to soil nutrients and water. In addition, the rhizobacterial IAA loosens the plant root cell wall and there by facilitates release of excess root exudates which could provide the rhizobacteria with additional nutrients [64]. Indeed, IAA production is dependent on the presence of enzymatic pathways in the bacteria being studied and concentration of tryptophan supplied in the media [65]. Moreover, the inherent ability of bacteria to produce IAA in the rhizosphere depends on the availability of precursor molecules and uptake of microbial produced IAA by plants [66].
These findings have a direct practical implication to enhance the growth, development and productivity of E. brucei for improved agroforestry practices in the southern and southwestern Ethiopia. Therefore, IAA producer strains from present study can be used in combination with the bacteria endowed with other phytobeneficial traits in the enhancement of the growth and development E. brucei and enrichment of its biomass with P for improved agroforestry practices.
Most of our phosphate solubilizing and IAA producer isolates are also HCN and NH3 producers. The 77.7% of the tested isolates exhibited HCN production. Nodule endophytic and/or rhizobacteria are well known for HCN production and this volatile secondary metabolite plays significant role in inhibiting growth of plant pathogenic fungi [67]. HCN produced by rhizobacteria are potent inhibitors of cytochrome C oxidase and metaloenzymes and hence affect the respiratory systems of the plant pathogenic fungi and result in fungal growth inhibition[67]. The existence of bacterial population in the soil with multiple PGP traits like phosphate solubilizing capacity, production of HCN, NH3 and IAA is well established[10, 11]. In addition, Rashid et al.[17] have reported several bacterial endophytes from different plant parts. These authors further indicated the presence of multifunctional traits like production of IAA, NH3, siderophore and hydrolytic enzymes in the endophytes.
Our isolates also exhibited the production of hydrolytic enzymes namely protease, lipase and chitinase. The 88.8%, 66.6% and 66.6% of our isolates exhibited lipase, protease and chitinase production, respectively (Table 4). Interestingly, isolate RG6 (Acinetobacter soli) is a good producer of all the evaluated plant growth promoting traits, though it is weak in production of lipase and protease. Amsalu et al. [8] have reported two protease and one lipase producing E. brucei root nodule bacterial endophytes. Production of different hydrolytic enzymes by bacterial endophytes is reported by several scholars[10, 11, 68]. In line to our findings, Prasad et al.[18] have also reported the synthesis of chitinase, protease or lipase by rhizobacteria. These authors have further indicated the involvement of these enzymes in the fungal cell wall and cell lyses. The root nodules of E. brucei are rich in phytobeneficial bacteria which can be used in the improvement and enhancement of growth and productivity of this multipurpose legume tree for improved agroforestry practices.
In applying the endophytes as bio-inoculants, it is equally important to consider their survival, adaptation and competitiveness in the rhizosphere soil conditions in the presence of indigenous soil bacteria. Therefore, in the present work, we also studied eco-physiological stress tolerance traits and carbon and nitrogen substrate utilization versatilities of the isolates endowed with the multiple plant growth promoting traits.
We report isolates that tolerated varied levels of eco-physiological stressors namely salt, pH, temperature and antibiotics. The 77.7% of our isolates exhibited growth between 0.5 and 3.5% salt concentrations in YEMA medium (Table 5). Salinity affects bacteria and host plant by inducing ionic stress through high concentration of ions and osmotic stress due to the change in osmotic concentration around cells causing desiccation and water deficit[70]. Our isolates that tolerated different levels of salt stress can be applied as bio-inoculants at saline soils to improve the growth and development this particular legume tree. In the present study, we also report E. brucei root nodule bacterial endophytes which can be applied as bio-inoculants in acidic and/or alkaline soils of Ethiopia to enhance the availability of P to the host plant grown in acidic and/or alkaline soils. The 66.6% of the evaluated isolates exhibited growth in pH ranges between 5.0 and 9.5. Therefore, the findings in this study have direct practical implication in Ethiopia, where 43% of soils are acidic [43]. As pH decreases, the bacterial cell structures like the import-export systems on the outer and inner membranes, periplasmic proteins, flagella, exo-polysaccharides, and cell walls may be adversely affected [71].
The identification and characterization of bacterial strains endowed with the multiple PGP traits should not be the only concern in the inoculants production and development. The development of inoculants which provide protection to themselves and their plant hosts against multiple eco-physiological stressors would be highly valuable for improved agro-forestry practices in the changing environmental conditions.
In this particular study, 33.3% of the isolates evaluated were grown in temperature ranges between 15 and 37°C, while 22.2% were managed to grow at 40°C. Higher temperature adversely affects symbiotic effectiveness of rhizobia and reduces host legume growth and development [70]. According to Hungriaet al. [71] the ability of rhizobial bacterial isolates to survive and grow at elevated temperature on pure culture media has strong correlation with their symbiotic performance under high temperature stressed soil conditions. Therefore, selection of high temperature tolerant rhizobia and/or other endophytic bacteria with plant growth promoting traits is very vital for selection as inoculants for tropical agriculture.
Isolates AU4 and RG6 (A. soli) exhibited tolerance to 83.3% of the antibiotics tested, while 55.5% of the isolates similarily exhibited tolerance to 66.6% of antibiotics. IAR test is vital in view of its importance to identification and taxonomic classification of bacterial strains [37, 71]. It is also one of the methods to screen for ecological competitiveness of the rhizobial strains in the soil [72].Therefore, bacteria endowed with such properties are good candidates for inoculants production. This is because the phytobeneficial root nodule inhabiting bacteria can be released into soil, when nodules are decomposed or disintegrated and lead saprophytic life style in the soil. In such soil conditions, antibiotic resistant bacteria can survive in soil environments. The multiple antibiotic resistances by root nodule endophytic bacteria in the present study could be attributed to previous exposure of these strains to the antibiotics tested in the soil as soil contains a lot of antibiotic producing microbes that coexist in the soil environment.
The 33.3% of our isolates have shown 100% carbon and 100% nitrogen source utilization, while 22.2% utilized 90% of the carbon and nitrogen substrates tested (Table 6). The bacterial versatility in utilizing a variety of carbon and nitrogen substrates has great practical implication for saprophytic life style of soil bacterial. Moreover, utilization of plant exudates carbohydrates by soil bacteria also indirectly contributes to phosphate mobilization by serving as a carbon source for phosphate solubilizing microorganisms [73]. Mayer and Merbach [74] have reported that treating P-deficient plants with IAA increased release of exudate carbohydrates by 52% implying that bacterial synthesized IAA could trigger high amount of plant carbohydrate exudates and provide better nutrient status of bacteria.