Characterization and Identification of Native Plant Growth-Promoting Bacteria Colonizing Tef (Eragrostis Tef) Rhizosphere During the Flowering Stage for A Production of Bio Inoculants

The rhizosphere is the region of soil that is affected by root secretions and soil associated microorganisms [1]. It is a nutrientrich region as compared to the bulk soil due to the accumulation of root exudates carrying a large number of carbohydrates and amino acids, serve as a rich source of energy and nutrients for rhizosphere microbes. Rhizosphere has been broadly classified into the three major zones.


Introduction
The rhizosphere is the region of soil that is affected by root secretions and soil associated microorganisms [1]. It is a nutrientrich region as compared to the bulk soil due to the accumulation of root exudates carrying a large number of carbohydrates and amino acids, serve as a rich source of energy and nutrients for rhizosphere microbes. Rhizosphere has been broadly classified into the three major zones. 3) Ectorhizosphere: that consists of soil closely adjacent to the root [2].
The rhizosphere is enriched with a variety of microorganisms and the bacteria residing in this region are called rhizobacteria [2]. Rhizobacteria are bacteria that inhabiting plant roots; they can multiply and colonize all the ecological niches found on the roots at all stages of plant growth, in the presence of a competing microflora in the rhizosphere [3]. Rhizobacteria can have a neutral, harmful or beneficial effect on plant growth, yield, and grain quality.
Harmful rhizobacteria are presumed to badly affect plant growth and development through the production of undesirable metabolic products. Beneficial rhizobacteria are termed as PGPR according to their mode of action. They are a group of free-living saprophytic bacteria that can be found in the rhizosphere in association with the root system and enhance plant growth and yield. It provides high adaptability in a wide variety of environments, faster growth rate, and a biochemical versatility that allows them to metabolize a wide range of natural and xenobiotic compounds. PGPR is characterized by the following properties (i) They must be able to colonize the root surface (ii) They must survive and multiply in rhizosphere associated with the root surface, in competition with other microorganisms and (iii) They must promote plant growth and control plants from phytopathogens [5].
PGPR directly affect plant growth by improving nutrient assimilation through fixation of atmospheric nitrogen, solubilization of phosphorus, siderophores production and/or other unavailable forms of nutrients. Production and/or suppression of growthregulating hormones (auxins, gibberellins, and cytokines) are another effective direct mechanism for plant growth enhancement [6]. Indirect mechanisms include competition for niche, production of antibiotic, production of lytic enzymes, ammonia production, HCN production, exopolysaccharide production and increased disease resistance ability of the plants [7]. It improves physicochemical and biological properties of the degraded substrates and makes them more suitable for plants with simultaneous increase of plant survival and seedling quality. Positive outcomes of PGPR application are an enhancement of seedlings emergence, faster plant growth, higher biomass production, an increase of root length, and branching, increased leaf area, and chlorophyll content [8].
PGPR species or strains also possess the enzyme ACC deaminase [9] and this enzyme can cleave the plant ethylene precursor ACC to ammonia and a-ketobutyrate thereby lowers the level of ethylene under various biotic and abiotic stress factors [10] such as salt stress, flooding stress, drought stress, heavy metal stress, and pathogen attack. Soil-borne pseudomonads fluorescent have excellent root colonizing capacity, catabolic versatility and produce a wide range of enzymes and metabolites that favor the plant withstand under varied biotic and abiotic stress conditions [11]. The improvement of plant growth can vary depending on colonization areas of PGPR on the host plant. It may inhibit the rhizosphere, the surface of the root, or even superficial intercellular spaces of the host plant [12].
The process of bacterial root colonization is under the influence of several mechanisms such as bacterial traits, root exudates production and several other biotic and abiotic stress factors [13]. Some authors reported that bacterial cells first inhabit the rhizosphere following inoculation into the soil [14]. Then, bacterial cells may occur as single cells attached to the root surfaces and consequently converted into doublets on the rhizodermis, forming a string of bacterial cells [15].
Bacterial colonization may then occur on the whole surface of plants and can even establish as micro-colonies (bio-films). It is important to note that the root system is not inhabited uniformly, different population densities being reported for the diverse root zones. For example, Kluyvera ascorbate inhabited the upper twothirds of the surface of canola roots, but no bacteria were detected around plant root tips [16]. Non-uniform bacterial colonization along the plant root can be explained by different factors such as varying root exudation patterns, bacterial quorum sensing effects as well as many other factors. The aim of this study is an isolate, characterize and identify potential PGP bacterial species or strains for production of bio inoculums serve as Biofertilizer and biocontrol to improve tef crops production and productivity without affecting soil fertility, living organisms, and their environment.

Study Area
This study was conducted in East Shewa Zone, Oromia Regional State, Ethiopia from 2009 to 2011 E.C. According to zonal statistics and information center, the zone is found between 38 o 57'and 39 o 32' E and 7 o 12' and 9 o 14'N and classified into three traditional agro-climatic zones [17]. The average altitude of the zone is 1,600 m but rises to 2,420 m at the northwestern and it falls to 900-1000 m towards the northeast [18] (Figure 1).

Soil Sample Collection and Analysis
71 composites soil samples were collected from the rhizosphere of cultivated tef varieties at a depth 0-20cm using an auger and these soil samples were dried, sieved through 2 mm sieve to have 1kg. The collected soil samples were then analyzed for selected physical and chemical properties like soil texture, pH, Electric Conductivity (EC), Organic Matter (OM), available phosphorus [19] and total nitrogen [20] at Debrezeit agricultural research center. A total of 426 (213 rhizosphere soil and 213 roots) samples were collected from the rhizosphere of cultivated tef varieties for microbial isolation.

Bacterial Isolation
For isolation of rhizosphere bacteria, 10gram of the soil was mixed in 90 ml of saline solution (0.9% NaCl) (w/v) to make a suspension. The prepared suspension was shacked for 5 minutes to remove unwanted substance such as soil, debris and dead bacterial cells from the samples. For isolation of root adhering bacteria, fresh roots were washed with distilled water to make a solution. Soil suspension and root washing solutions were serially diluted (from 10-1 to 10-6) respectively. 0.1mL of supernatant from each dilution of rhizosphere soil and root washing solution was transferred onto prepared solid medium and incubated at the appropriate temperature. All the bacterial isolates were morphologically characterized as per the method described in Berge's manual of determinative bacteriology and in vitro evaluated for different bacterial plant growth-promoting traits, biocontrol properties, and abiotic stress factors tolerance activities.

Characterization of Bacterial Isolates for their Plant Growth-Promoting Traits
All the isolates were characterized for bacterial PGP traits such as nitrogen fixation, phosphate solubilization, organic acid production, Indole Acetic Acid (IAA) production.
Test for Phosphate Solubilization Activity: Bacterial isolates were spot inoculated on the Pikovskay's agar medium plates and incubated at 30°C for 5 days. The appearance of the clear zone around bacterial growth was used as an indicator for positive bacterial phosphate solubilization activities [22].

Test for Organic Acid Production:
The bacterial organic acid production test was conducted by inoculating test bacterial culture in minimal salt medium broth for 2 to 3days at 30 o C. The appearance of pink color in the medium indicates a positive result for bacterial organic acid production using methyl red as an indicator [23].
Production of Indole Acetic Acid (IAA): IAA production was detected as described by [24]. Bacterial cultures were grown on

Characterization of Bacterial Isolates for their Biocontrol Properties
All the rhizobacterial isolates were characterized for phytopathogens controlling properties such as ammonia production, hydrogen cyanide production, hydrolytic enzymes production, and exopolysaccharide production.
Production of ammonia. Bacterial isolates were tested to produce ammonia in peptone water described by Kumar et al. [25]. Freshly grown rhizobacterial isolates were inoculated in the tubes containing 10ml peptone water and incubated for 48 h at

Hydrogen Cyanide (HCN) Production Test
Bacterial isolates were tested for HCN production by the methodology described by Castric [26]. The isolates were

Hydrolytic Enzyme Production Test
Rhizobacterial isolates were tested for their hydrolytic enzyme production like protease, cellulase, amylase, and pectinase.

Protease Production Test
Bacterial isolates were tested for their ability to produce proteolytic enzymes onto skim milk agar 3% v/v) medium [27,28].
The diameter of the clear zone formed around the growing bacterial colonies was measured after 48 h of incubation at 30°C.
Bacterial isolates showing clear zone on the proteolytic medium was indicated a positive result for protease production.

Cellulase Production Test
Pure cultures of bacterial isolates were individually transferred onto CMC agar plates. After incubation for 48 hours, CMC agar plates were flooded with 1% Congo red and allowed to stand for 15 min at room temperature. One molar NaCl was thoroughly used for counterstaining the plates. Clear zones were appeared around growing bacterial colonies after 48 hours of incubation at 30 o C indicating bacterial cellulose production [29].

Amylase Production Test
The bacterial isolates were spot inoculated on starch agar medium plates and incubated at 30°C for 48 h. At the end of the incubation period, the plates were flooded with iodine solution, kept for a minute and then poured off. Iodine reacts with starch to form a blue color compound. This blue color fades rapidly. Hence the colorless zone surrounding bacterial colonies indicates bacterial ability to produce amylase [30].

Pectinase Production Test
The rhizobacterial isolates were spot inoculated on the pectin agar plates and incubated for one week at 30 o C. The plates then were flooded with 1% iodine solution for one hour, drained, rinsed with water and observed halo zone formation around growth colonies indicating bacterial pectinase production [31].

Exopolysaccharide (EPS) Production
Rhizobacterial isolates were tested for EPS production ability using solid medium such as Burk's medium (g/1: Sucrose, 20.0; isolates were extracted and confirmed for exopolysaccharide production using cold acetone through observation of precipitate formation [32].

Characterization of Bacterial Isolates for Abiotic Stress Tolerance Ability
Stress tolerance test was carried out by growing the bacterial isolates at different abiotic stress factors such as temperature, pH, and salinity. Temperature is one of the abiotic stress factors that affect plant growth and yield. Temperature effects on PGP bacterial isolates were studied by streaking the isolates on nutrient agar plates and incubating at 4, 20, 30, 40, 50 and 60°C for 48 h and observed for colonial growth on agar medium. pH is another stress factor that affects plant growth, for this rhizobacterial isolates were transferred on to nutrient agar with pH 4, 5, 7, 9, 11 and 13 was prepared using H2SO4 and KOH. Bacterial isolates were streaked on a different medium and grown at 30°C. Likewise, for salinity stress nutrient agar medium with 0.86, 1.7, 2.3 and 3.4 M NaCl (5%, 10%, 15% and 20% NaCl) was prepared and the isolates were grown on the medium at 30°C and observe their growth on agar medium.

Biochemical Characterization and Identification of PGP Bacteria
Biochemical bacterial characterizations were carried out using the procedures described in the Bergey's Manual of Systematic Bacteriology. Morphological identification of PGPR isolates was performed using morphological characterization. After 24 h of bacterial isolates growth on peptone agar at 30°C and characterized for their following traits i.e. color, shape, size, surface, opacity, and texture. Cellular morphology such as color and shape and also division mode was identified by using light microscopy. Carbon source utilization patterns of rhizobacterial isolates having plant growth-promoting traits, biocontrol and stress tolerance properties were analyzed using Biolog OmniLog ® ID System which is now widely available to assess functional diversity of bacteria and fungi in the rhizosphere. Biolog is patented technology and automated/ semi-automated biochemical and physiological tests that analyze microbial ability to utilize particular carbon sources, and chemical sensitivity assays.
The Biolog OmniLog identification system is that characterizes the ability of microorganisms to utilize or oxidize (simply "Biolog" Inc, Hayward, California), a system that used Gene III microplate containing 94 phenotypic panels of 71 carbon sources assays and

Seed Germination and Seedling Growth Test
Seed germination tests were carried out to determine the effect of inoculated rhizobacterial species or strains on the rates of seed germination and seedling growth [32]. For this, tef seeds were used as plant materials. Healthy seeds were surface sterilized with 70% alcohol for 3 min and followed with 1% hypochlorite for 5 minutes and rinsed 5 times with sterile distilled water. Four PGPR species or strains (single and consortium) were grown in nutrient broth on shaking incubator (180rpm) at 30°C for 24 h. The surface-sterilized seeds of tef were inoculated in broth culture of the PGPR species or strain cultures for 30 min including normal water (C) as control. 25 inoculated seeds of each treatment were placed in separate Petriplate containing soaked filter papers and the Petri-plates were incubated at room temperature for 7 days. Seed germination was recorded regularly starting from the 2nd day based on the number of the germinated seed out of total germination.
Each treatment was replicated three times. Percentages of germination and seedling growth were calculated and evaluated the effect of single and consortium-seed bacterization on a shoot and root growth of tef crops. Vigor index of seedling was measured on the last day of the experiment according to the formula proposed by Abul-Baki & Anderson [33]. Seed vigor index = (mean shoot length + mean root length) × germination%.

Methods of Data Analysis
Data analysis was carried out using the chart, table, frequency, and percentiles using excel to evaluate PGP traits, biocontrol properties, and abiotic stress tolerance of native plant growthpromoting rhizobacteria.

Soil Physiochemical Analysis
71 composites soil samples were collected from a depth of 0-20cm using auger and these soil samples were dried, sieved through 2 mm sieve to have 1kg. The collected soil samples were then analyzed for selected physical and chemical properties mainly soil texture, pH, organic matter, organic carbon, total nitrogen, available phosphate, and electrical conductivity. The majority of the soil samples collected from the study area was found to be in the alkaline pH range [34]. 73.4 % of soil samples were found to be in the lower range of total nitrogen, 26.6% soil samples were found to be in the medium range of total nitrogen. 66.7% of soil samples were found to be in the lower range of available phosphate and 26.3% of were found to be in the medium range of available phosphate and 7% were found to be in the higher range of available phosphate. For availability of the organic matter, 90.7% of soil sample was found to be in the lower range and 9.3% were found to be in the medium range of organic matter.
For electrical conductivity were the majority of the experimental soil samples its electrical conductivity was found to be at the acceptable range for crop production (Table 1).

Characterization of Bacterial Isolates for Plant Growth-Promoting Traits
457 pure bacterial isolates associated with tef rhizosphere were characterized for different PGP traits such as phosphate solubilization, organic acid production, IAA production, growth on nitrogen-free medium and NH3 production. Among these, 71.3% (326) rhizobacterial isolates were positive for phosphate solubilization test, 57.1% (261) isolates were positive for IAA production, 52% (238) isolates were positive for organic acid production and 12% (55) positive for ammonia production and 10% (46) bacterial isolates were grown on nitrogen-free medium ( Table 2).   (Table 3).

Characterization of Bacterial Isolates for Abiotic Stress Factors
All bacterial isolates were characterized for abiotic stress factors tolerance activities such as (5% NaCl, 10% NaCl, 15% NaCl and 20% NaCl w/v), (

Seed Germination and Seedling Growth Test
Seed germination and seedling growth test was performed  for solubilization of mineral phosphates [37].
Soil pH can also play a role in Nitrogen volatilization losses.
Ammonium in the soil solution exists in equilibrium with ammonia gas (NH3). The equilibrium is strongly pH-dependent. If NH4+ were applied to the soil at pH seven, the equilibrium condition would be 99 % NH4+ and 1% NH3. At pH 8, approximately 10 % would exist as NH3 gas. This means that a fertilizer like urea is generally subject to slightly higher volatilization losses at a higher soil pH. Use of nitrogen-fixing plant growth-promoting rhizobacterial inoculant as biofertilizers is one of the best alternative comparing to the chemical treatment for improving plant growth and its productivity through the production of complex nitrogenous enzymes. Nitrogen fixation ability is an important criterion for the selection of potential PGPR.
In this study, 10 % of bacterial isolates were grown on N-free from the rhizosphere are more efficient IAA producers than isolates from the non-rhizosphere soil. This is an important mechanism of plant growth promotion because IAA promotes root development and uptake of nutrients [41].
It has long been proposed that IAA act coordinate demand and acquisition of nitrogen and enhance crop yields. In this regard, Patten and Glick showed that the production of IAA by Pseudomonas putida was closely linked to the optimal development of the root system in Brassica napus L. and Vigna radiata [42].
Another important trait of PGPR in the production of ammonia that indirectly influences plant growth. In this study, 12 % of the isolates were able to produce ammonia, which is an inorganic volatile, may be useful in biocontrol as demonstrated by Howell et al. [43] where ammonia, produced by PGP bacterial species, appeared to be one of the many mechanisms that bacteria use in the biocontrol of preemergence damping-off caused by fungal pathogens. The presence of ammonia producing PGP bacteria is indicative for PGP bacterial biocontrol properties were takes place in the rhizosphere than nonrhizosphere soil. Hydrolytic enzymes act as agents for prevention of plant diseases by causing lysis of pathogenic microbes in the close vicinity of the plant as they secrete increased level of cell wall lytic enzymes (chitinase, glucanase, lipase, and proteases) [44]. In this study, 65.4 % isolates were positive for protease production, 20.6 % isolates were positive for amylase production, 8% isolates were positive for cellulase production, and 0.3% isolates were positive for pectinase production. PGPR that synthesizes one or more of these lytic enzymes has been found to have the ability to control a range of plant pathogenic fungi and bacteria and enhance crop productivity. Bull et al. [45] who reported that Lysobacter and Myxobacteria produces lytic enzymes have shown effectiveness against some plant pathogenic fungi.
HCN production by rhizobacteria has been postulated to play an important role in the biological control of pathogens [46]. In this study, 15.4% of the bacterial isolates were positive for HCN production, which acts as an inducer of plant resistance. Several factors have been reported to influence the rate of HCN production.
Glycine is the direct precursor of microbial cyanide production and it has been found in root exudates [47]. HCN secreted by P. fluorescent strain has been demonstrated to stimulate root hair formation and suppress back root rot caused by Thielaviopsis basicola in tobacco plant [48]. Exopolysaccharide is the active constituents of  (6) bioremediation [49]. In this study, 18.6% of bacterial isolates were producing EPS. EPS-producing PGPR can significantly enhance the volume of soil macropores and the rhizosphere soil aggregation, resulting in increased water and fertilizer availability to the applied plants [50].
Exopolysaccharide protects beneficial rhizosphere competing bacteria from various environmental stresses [51], protects cells from antimicrobial compounds, and antibodies, or for sticking to other bacteria, animal and plant tissues. Abiotic stresses are major factors that limit crop production and are the cause of more than 30% of worldwide losses [52]. Abiotic stress factors include temperature, pH, salinity and heavy metal contamination.
Importantly, even though many plant growth-promoting bacteria associated with plant rhizosphere show good results during in vitro evaluation and they fail in the field when applied as plant growthpromoting abiotic stress tolerating bacterial inoculants. One main reason for their failure is the stress imposed on them by the sudden change of soil physical, chemical and biological matters in the environment [53].
Characterization of PGP bacteria for stress tolerance ability is an important factor in the selection of potential bacterial species for the development of PGP bacterial inoculants to sustain agricultural production and productivity. Salinity is one of the most important abiotic stress factors that undesirably affect plant growth and productivity in the world. It is one of the global problems that affect agricultural productivity and production: about 20 % of cultivated This result showed that isolates were survived at wide range temperatures and improve plant growth and yield.
PGP rhizobacterial species or strains can produce the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase that breaks down ACC, the direct precursor of ethylene, into ammonia and a-ketobutyrate and support the plants for vigorous growth and development at environmental conditions. For seed germination and seedling growth status evaluation, a seed of tef was inoculated with 4 identified PGP bacterial species having excellent PGP traits.
Seed coating was made by using single and consortium bacterial inoculation system. All of the tef seeds inoculated with potential PGP bacterial species or strains were germinate tef seeds up 100% on 3rd and 4th days after inoculation and increase mean shoot length and roots length of the inoculated seeds up to 3 and 2.6 cm respectively in comparison to control (Table 6). Vigor index of seedling was measured from380 to 550 on the last day of the experiment. Pradhan [55], he reported that seeds inoculated with Bacillus sp. were significantly increased the germination percentage, root and shoot length of the crops as compared to the untreated one. Besides, Pieterse and Van Loon [56] reported that 30% growth improvement of Arabidopsis accession was grown in autoclaved soil due to inoculation with Pseudomonas fluorescens. According Woyessa and Assefa [57] report inoculation of tef crops with Pseudomonas fluorescent identified from tef rhizosphere increases mean root dry weight up to 39%, root shoot ratio up to 42%, and grain yield up to 28% and also tef crops inoculated with Bacillus subtilis that increase mean root dry weight of tef up to 28%, root shoot ratio up to 19% and grain yield up to 44%.

Conclusion and Recommendation
At a global scale, the effects of continuous and heavy use