Isolation and Characterization of Efficient Phosphate Solubilizing Bacillus (PSB) from Different Agro-ecological Zones of Tigray Soil, Ethiopia

Applications of biofertilizer have great practical importance for increasing fertility of the soil and reducing environmental pollution. Screening and characterizing phosphate solubilizing Bacillus (PSB) strains from different agroecologies of Tigray soil and in vitro assessment for the adaptability under different abiotic stress would help in selecting the most efficient strain for use as biofertilizer. A total of 64 soil samples were collected from different agro-ecological zones of Tigray and checked for the presence, capability and efficiency of PSB. Out of 64 soil samples 21 of them were phosphate solubilizing. These phosphate solubilizing strains again incubate at 37 ̊C for 48h on pikovskaya medium to see efficacy of phosphate solubilization. Highest efficiency was recorded in MUB28, MUB47and MUB64 isolates and also tolerant to acidity and alkaline up to pH4 and pH8, respectively. And difference was recorded in saline tolerance among the strains: MUB47 is the most saline tolerance strain which tolerate saline up to 513mM of sodium chloride (NaCl) but MUB28 and MUB64 have tolerated saline up to 340mM of NaCl. The identification of efficient PSB candidate strains with salt & pH tolerant features in the soil sample could have an implication on the end-users to obtain the desired beneficial effect such as maintaining soil healthy, provision of adequate phosphorus from soil to the plant and improving crop yield of Tigray Agriculture. Based on the morphological, microscopic, biochemical and physiological characterization, it may be inferred that the diversity among these efficient PSB strains is low.


INTRODUCTION
The demand for increasing productivity necessitates provision of adequate nutrients from the soil. However, most agricultural soils of tropics, including Ethiopia are deficient in sufficient phosphorus, besides nitrogen. It is reported that the two nutrients often limit crop production in Ethiopia (Dibabe, 2000).
Tigray, the most northern region of Ethiopia is generally regarded as the most degraded part of the country. This is manifested in the form of soil erosion, deforestation, declining bio-diversity resources, and soil moisture stress (Taffere, 2003). The leading economic sector of the region is agriculture over 85% of the populations are farmers who depends on the high input demanding © CNCS, Mekelle University 263 ISSN: 2220-184X varieties and chemical fertilizers in order to improve crop yield (Edwards and Belay, 2003).
However, the cost of these inputs is beyond the purchasing power of most farmers. In addition, prolonged use of chemical fertilizers has resulted in a number of negative side effects on the environment (Edwards and Belay, 2003). Long-term application of chemical fertilizer has caused some plant nutrients to be depleted and other to be deposited in excess in the soil, and consequently the acidity of the soil increased (Reddy et al., 1991). Hence, searching alternative strategies to improve soil health and crop yield without causing damage to the soil and environment is a focus of researchers for a number of years.
Biofertilizers are, therefore, gaining importance as they are eco-friendly, nonhazardous and nontoxic (Sharma et al., 2007). Biofertilizer refers to products consisting of selected and beneficial living microbes, which are added to soil as microbial inoculants. These microbes are known to improve plant health by secretion of growth promoting substances and by increasing the availability of micronutrients (Sharma et al., 2007). Inoculation of phosphate solubilizing Bacillus (PSB) results in simultaneous increases in crop yields and phosphorous uptake (Datta et al., 1982). Reports also indicated that Bacillus inoculants are increasing wheat yield up to 43% (Kloepper et al., 1989).
Phosphate solubilizing Bacillus (PSB) can be isolated from soils of different environmental conditions. It is necessary to characterize its efficiency in terms of phosphate solubilizing ability before PSBis used as biofertilizer. PSB can also be characterized under different morphological, physiological and biochemical traits to see their adaptability for different agroecologies and diversity. This would help in selecting the most efficient strain for use as biofertilizer. This study is, therefore, aiming at isolating and characterizing efficient phosphate solubilizing Bacillus from different agroecologies of Tigray soil and assesses their suitability. traditional: farmers ploughing their fields by a pair oxen using the local plough called "Mahresha", sow and harvest by hand, production increases resulted from expanding cultivated area and not from increasing yield (Taffere, 2003). The agroecological zones and niches of Tigray are diversified with distinct vegetation cover, soil and other natural resources.

Study Design
PSB was isolated from soils of different environmental conditions and selects the most efficient strain to recommend for the use of bio-fertilizer. Further the isolates were characterized under different morphological, physiological and biochemical traits to see their adaptability for different agroecologies and diversity of the isolates.

Soil Sample collection
A total of 64 soil samples were collected from different agro-ecology zones of Tigray. The soil samples were collected from the surface of (0-30cm) several spots of each sampling sites and mixed (homogenized). The composite soil samples were then sub-sampled and transported in sterilized polytlene bags to Mekelle University, Department of Biology, Microbiology laboratory.

Isolation of phosphate solubilizing Bacillus (PSB)
Bacillus strains were isolated from the soil using a heat treatment at 100°C during 25-30 min.
One gram of soil from each sample was suspended in 9 ml of sterilized water to make 1:10 dilution. These were shaken on shaker for 30 minutes to break clogs. Then series of dilution were made (10 -4 -10 -9 ). Pikovskaya medium which was autoclaved at121 0 C and pressure 15psi for 15 minutes was used to isolate PSB. From the diluted soil sample 0.1ml of suspensions were transferred on Petri dishes containing 15 ml pikovskaya medium. The suspensions were spread uniformly on Petri dishes using glass rod spreader and incubated for 5 days at 30±2˚C. Colonies showing clear zone around them were picked and subculture for purification (Pikovskaya, 1948).

Solubilizing efficiency
All phosphate solubilizing Bacillus were spot inoculated on pikovskaya medium and incubated at 37˚C for 48 h for detection of their efficacy. Halos surrounding the colonies were measured and the solubilizing efficiency (SE) was calculated as follow (Pikovskaya, 1948): Solubilizing efficiency (SE) = Solubilization diameter X 100(Equation -1) Growth diameter

Morphological and microscopic characterization of efficient PSB strains
Colonies were characterized on the basis of color, elevation, shape, margin and surface of the colonies. Simple staining was performed to determine cell shape and cellular arrangement. A culture of 24 and 48 hour old efficient PSB strains were gram stained and spore stained, respectively (Nanda, 2005;Prescott et al., 1999).

2.7.Motility test
The motility test determines the presence of flagella, external appendages used by the efficient PSB for movement. An isolate of PSB was inoculated in a tube containing Sulfide, Indole, Motility (SIM) medium with sterile transfer needle. The needle was inserted and with drawn in a straight line in the center of the medium. It was incubated for 48 hours at 30-32°C. Growth away from the line of inoculation indicates that the organism is motile. PSB without flagella do not spread away from the line of inoculation so they are non-motile (Michael and Burton, 2011).

Starch hydrolysis
Starch agar (20 ml soluble starch (10%), 100 ml melted nutrient agar) was inoculated with young culture of efficient PSB strains and incubated at 32 0 C for 3 -5 days. The culture was then flooded with Lugol`s iodine solution. The enzymatic hydrolysis of starch by amylases was indicated by clear zone around the colony. Unchanged starch gave a blue color (Collins and Lyne, 1970).

Catalase test
Catalase activity was checked by flooding fresh 3% hydrogen peroxide on to 18-24 hour old efficient PSB culture on agar slant the enzymatic hydrolysis of hydrogen peroxide was indicated by bubbles of oxygen, for non-catalase producing bacteria no oxygen bubbles are observed (Michael and Burton, 2011).

Citrate utilization test
Simmon citrate agar was streak plated with a young efficient PSB culture and incubates at 32 0 C for 24 hours. Utilization of citrate was indicating blue color in the medium (Michael and Burton, 2011).

Methyl red test (MR test)
Test tubes containing 5 ml MRVP broths were inoculated with 24 hours old efficient PSB culture and incubate at 30±2 0 C for 2-3 days. Then 5 drops of methyl red indicator were added and gently rolled between the palms to disperse the methyl red. The development of red color indicates a positive reaction (Pacarynuk and Danyk, 2005).

Vogues proskauer test (VP test)
A test tube containing 5 ml MRVP broths were inoculated with 24 hours old efficient PSB cultures and incubate at 30±2 0 C for 2-3 days. Then one ml of VP reagent-1 (6g of alpha naphtol dissolved in 100ml of 96% ethanol) and one ml of VP reagent-2 (16g KOH dissolved in 100ml double distilled water) were added to the MRVP broth respectively. The formation of pink or red color represents positive VP test or acetone production. A yellow or brown color is a negative result (Pacarynuk and Danyk, 2005).

Urea test
Test tubes containing 5ml urea broth were inoculated with efficient PSB culture and incubate for 48 hours at 35 0 C. If ammonium is produced as a result of urea hydrolysis, the increase in pH will develop pink colour (Michael and Burton, 2011).

Indole production test
Test tubes containing 5ml SIM medium were inoculated with efficient PSB culture and incubate for 48 hours at 30-32 0 C. Subsequently five drops of kovac`s reagent was added. The test tubes were allowed to stand. The development of a red color in the top layer will indicate a positive result for indole production and no red color development represents a negative test (Michael and Burton, 2011).

Hydrogen sulphide (H 2 S) test
Test tubes containing 5ml SIM medium were inoculated with efficient PSB culture and incubate for 48 hours at 30-32 0 C. When H 2 S is produced, the strip turns from brown to black (Rangaswami and Bagyaraj, 2005).

Salt tolerance
Three strains PSB1, 2 and 6 had significantly increased the germination percentage of the tomato seeds at salt concentration between 0 to 60 mM (Anjali et al, 2013

Data analysis
Morphological, microscopic, biochemical and physiological character was used to determine the matrix similarity index of the efficient PSB strains using Adansonian method (Sneath and Sokal, 1962).

(Equation -2)
Where Ns is the number of similar (positive) characteristics and Nd is the number of different characteristics (positive in one and negative in the other).

RESULTS AND DISCUSSION
A total of 64 soil samples were obtained from different agro-ecological zones of Tigray. These soil samples were checked for the presence of efficient PSB. It was found that 21 of them were able to form clear zone on pikovskaya medium which indicated that 33% of them were phosphate solubilizing (particularly, calcium phosphate solubilizing) and again 14% of the PSB(3 out of the 21 PSB) were efficient in phosphate solubilizing ability i.e. MUB28, MUB47, MUB64 (Table 1). The specific places where MUB28, MUB47, MUB64 isolates found are "Adigolagul" at an altitude of 2527 m above sea level (asl), "Maydeleata" at an altitude of 1896 m (asl) and "Duhutsa" at an altitude of 2289 m asl, respectively (Fig 1), which are high and moderate land areas of Tigray (Taffere, 2003).
Soil inoculation with phosphate solubilizing bacteria showed improvement in solubilization of fixed soil phosphorus and resulted in higher crop yield (Freitas et al., 1997, Nautiyal, 1999. It could be then inferred that these efficient isolates were highly promising biofertilizers in increasing agricultural productivity.  These efficient PSB strains were further subjected to microscopic, morphological, biochemical and physiological characterization: all the strains were Gram positive, spore forming, rod shaped with circular end and their cellular arrangement was single. The morphological characteristics also clearly indicated that all the strains have circular shape, mucoid surface, flat elevation and with entire margin (Table 2). Citrate and starch were utilized as source of carbon. Moreover, difference among the isolates in production of catalase was recorded. The result showed that MUB28 and MUB64 were catalase positive, and MUB47 was catalase negative (Table 3). Therefore, MUB28, MUB47 and MUB64 were evaluated in vitro for their ability to tolerate salt and pH (Table 4). Additionally, the pH values of the soil sample from the different agro-ecological zones of Tigray were measured and it was within the range of 5.5 -7.5. In vitro the efficient PSB strains had a tolerance to acidity and alkalinity beyond the P H range of the environment (5.5 -7.5). MUB28, MUB64, MUB64 were tolerant to acidity and alkaline up to pH4 and pH8, this makes them highly tolerant to acidic and alkaline environment of Tigray.
Thus, these kind of acid and alkaline pH tolerant strains of PSB will have paramount importance as biofertilizers in Tigray. Difference was also recorded in salinity tolerance among the strains. MUB47 is the most saline tolerance strain which tolerate saline up to 513mM of NaCl but MUB28 and MUB64 had an ability to tolerate saline up to 340mMof NaCl (Table 4). High salinity tolerance organisms can be used for preparation of biofertilizer (Rajankar et al., 2007). Hence the strains could be ideal as phosphate bio-fertilizer. Organisms with a high similarity index, eighty percent (80%) or more, are grouped together (Sneath and Sokal, 1962). The matrix of similarity index of these efficient phosphate solubilizing Bacillus strains was more than 80% (Table 5), and this was high similarity index, making it difficult to differentiate them. So, it may be inferred that the diversity among the groups is low.

CONCLUSION
Phosphate solubilizing Bacillus (PSB) can be isolated from soils of different environmental conditions. It is necessary to characterize its efficiency in terms of phosphate solubilizing ability before PSB is used as biofertilizer. Out of 64 soil samples 33% of them were phosphate solubilizing this indicates that PSB is common in Tigray soil. Moreover, highest efficiency was recorded in MUB28, MUB47and MUB64 isolates and also tolerant to acidity and alkaline up to pH4 and pH8, respectively. And difference was recorded in saline tolerance among the strains: MUB47 is the most saline tolerance strain which tolerate saline up to 513 mM of sodium chloride (NaCl) but MUB28 and MUB64 have tolerated saline up to 340 mM of NaCl. These efficient PSB strains showed high similarity index above 80%, it may be inferred that the diversity among these efficient PSB strains is low. The identification of efficient PSB candidate strains with salt & pH tolerant features have an implication on the end-users to obtain the desired beneficial effect such as maintaining soil health, provision of adequate phosphorus from soil to the plant and improving crop yield of Tigray Agriculture. The presence of such efficient PSB strains suggesting to enlist them as candidate strains for further characterization, and application under greenhouse and field condition. This study therefore paves the way for ideal selection of efficient PSB strains with desired features under field condition so as to increase the agricultural productivity of the region.

ACKNOWLEDGEMENTS
This work gets fund award from Mekelle University, College of Natural and Computational Sciences. We also like to say thank you to Mr. Daniel Gebremeskel for his help in developing MAP of sampling sites.