Effect of organic and inorganic fertilizer applications on phosphate solubilizing bacteria in the rhizosphere of maize (Zea mays L.)

A field experiment was conducted between August and October, 2014 at the Department of Microbiology to assess the effect of organic and inorganic fertilizers on the population and activities of Phosphate Solubilizing Bacteria (PSB) in the rhizosphere of maize variety BR9928DMRSR-Y. Three treatment groups were used in the study and these were: Groups which received applications of organic fertilizer (Poultry litter) alone, groups which received applications of inorganic fertilizer (NPK) and the third group which was the control (CON) did not receive any fertilizer application. A total of twenty-three bacteria were isolated and in-vitro screening was done for different phosphate solubilization activity. The study revealed that maximum population of total heterotrophic bacteria (26.8×109 CFU/g) was obtained with organic fertilizer (OM) treatment. Among the different PSB isolates, OMPSB had the highest bacterial count of 10.2×109. The lowest bacterial populations were obtained from IFPSB with 6.0×109 CFU/g. Out of the 23 PSB isolates, 18 were positive for phosphate solubilization with OMPSB8 showing the highest zone with 16 mm. Results showed that application of organic fertilizers enhanced the bacterial population and also showed increase in phosphate solubilization activities in rhizosphere soil compared to NPK and control treatments. This shows that organic fertilizers would be able to sustain the soil fertility for a longer period by meeting the demand of present and future generations. 
 
 Key words: Treatment groups, population, phosphate solubilization, in-vitro screening


INTRODUCTON
Soil nutrient depletion has been a major challenge in Nigeria as a result of continuous cultivation of soils without adequate addition of external inputs. This decline may occur through leaching, soil erosion and crop harvesting (Muchena et al., 2005;Mutegi et al., 2008).
Soil nutrients face the risk of continuous decline unless the nutrients are replenished through the use of organic or mineral fertilizers, partially returned through crop residues or through traditional fallow systems. The continued soil nutrient depletion has a negative impact on *Corresponding author. E-mail: patrickrichard.ok@gmail.com. Tel: 08063853332.
Author(s) agree that this article remains permanently open access under the terms of the Creativ e Commons Attribution License 4.0 International LicenseF sustainable agriculture as crop production especially that of maize is decreasing (Hussain et al., 2007). Maize (Zea mays L.), an annual crop belonging to the grass family Poaceae (OECD, 2003;USDA, 2005), together with rice and wheat are the three most important cereal crops in the world (Min et al., 2015). Maize has the following advantages compared to other cereals: High production, easy processing, easy digestibility, and they are less expensive. It is an important source of carbohydrate, protein, iron, vitamin B and minerals (IITA, 2009) which according to IITA (2001) report, contains 80% carbohydrate, 10% protein, 3.5% fiber and 2% mineral. Thus, it is very essential for food and can also be used as forage and feed for livestock because it is among the highest in net energy content and lowest in protein and fibre content (Oladejo and Adetunji, 2012).
In order to maximize the yield of maize crop, improved cultural practices such as organic and inorganic (chemical) fertilizers application can be used.
Though, application of chemical fertilizers can improve the nutrient balance of soils, which may lead to increases in crop yields, its continuous use is hazardous both to human health and the environment (Glick, 2003). This may cause plant toxicity (Nazar et al., 2012) and the bioaccumulation of trace metals in plants may pose a health risk consumed (Khan et al., 2015;Roy and McDonald, 2015). However, the negative effects of chemical fertilizers can be avoided by using organic fertilizers which have a positive effect on the Plant Growth Promoting Rhizobacteria (PGPR) -a group of rhizosphere bacteria found in association with roots which can enhance the growth of plant directly or indirectly by helping plants in nutrient uptake from rhizospheric soil (Mia et al., 2010). Organic fertilizers also play key role in sustaining soil fertility and crop productivity (Soumare et al., 2003;García-Orenes et al., 2016).
Furthermore, to increase the natural fertility of the soil and develop new approaches to reduce the need for chemical fertilizers, PGPR are recognized as important factors in sustainable agricultural production as they may be important for plant nutrition by increasing the P uptake by plants, playing a significant role in the bio-fertilization of crops (Idriss et al., 2007) and serving as a natural source of fertilizers that improve the efficiency of soil and plants (Khalid et al., 2004). The rhizosphere supports large and active microbial population such as bacteria, fungi, nematodes, protozoa, algae and microarthrops (Raaijmakers and Weller, 2001) which play important roles in ecological fitness of their plant host (Kent and Triplett, 2002).
Diverse PGPR strains have been used successfully for crop inoculations and to enhance plant growth (Kumar et al., 2012); these comprise members of the bacterial genera Azospirillum (Cassán and García, 2008), Bacillus (Jacobsen et al., 2004), Pseudomonas (Loper and Gross, 2007), Rhizobium (Long, 2001), Serratia (De Vleeschauwer and Höfte, 2007), Stenotrophomonas (Ryan et al., 2009) and Streptomyces (Schrey and Tarkka, 2008). Pseudomonas and Bacillus genera are the most commonly investigated PGPR, and often the dominating bacterial groups in the rhizosphere (Morgan et al., 2005). The modes of action of PGPR involve complex mechanisms to promote plant growth, development and protection through phytostimulation, bio-fertilization and biocontrol (Bloemberg and Lugtenberg, 2001). Some of these bacterial species have mineralization and solubilization potential for organic and inorganic phosphorus respectively (Hilda and Fraga, 2000;Khiari and Parent, 2005). Phosphate rock minerals are often too insoluble to provide sufficient P for crop uptake; therefore, the use of phosphate solubilizing bacteria (PSB) can increase crop yields up to 70% (Verma, 1993). The aim of this work was to determine the effect of organic and inorganic fertilizer on the phosphate solubilizing bacteria present in maize plant rhizosphere. In a previous study by Azani (1995), chicken dung showed the best fertilizer compared to cow dung and NPK, thus, chicken dung was chosen as phosphorus source in this study.

Experim ental design
The experimental design used for the field experiment w as the randomized complete block design as described by Kuntal et al. (2007). The experimental field w as sited behind the Department of Microbiology, University of Ibadan, Ibadan, Oyo State. The field w as divided into six blocks; tw o w ere treated w ith organic amendments (poultry litter), tw o w ith inorganic fertilizer (N:P:K 12:12:17), and tw o w ithout any treatment and w as considered as the control. Grains of maize variety BR9928DMRSR-Y w ere obtained from IITA, Ibadan headquarters and these w ere sow n by planting three seeds/hole in each block. A total of ten holes w ere bore in each block.

Sam pling and analysis
Sampling w as done for a period of 56 days. Rhizosphere soils w ere collected at different grow th stages of the maize (14, 28, 42 and 56 days after planting), by uprooting four plants from each treatment and keeping the soil around root system intact. After removing the bits of plant roots and other debris, the soil particles w hich adhered strongly to the roots w as immediately used for analysis w ithout drying follow ing the method described by Basul et al. (2010). During the sampling period, population of Total Heterotrophic Bacteria (THB) and Phosphate Solubilizing Bacteria (PSB) in rhizosphere soil w as counted at 14 days interval (that is, 14, 28, 42 and 56).

Isolation and enum eration of rhizosphere bacteria
Three replicate samples of rhizosphere soil w ere taken for enumeration of total heterotrophic count. The serial dilution plate technique as described by Johnson and Curl (1972) w as employed to enumerate the rhizosphere soil by plating on nutrient agar using \ the pour plate method. The plates w ere incubated at 37°C for 24 h. After the incubation period, the colony forming units w ere counted and expressed as colony forming unit per gram (CFU/g) of soil. Phosphate solubilizing bacteria w ere isolated by inoculating into sterile Petri dishes of Pikovskaya medium (Pikovskaya, 1948). The plates w ere incubated at 37°C for 48 h. After the incubation period, the colony forming units w ere counted and expressed as CFU/g of soil. Colonies w ith a 'halo' on the different media w ere sub-cultured onto nutrient agar plates. The phosphate solubilizing bacteria w ere isolated using Pikovskaya medium (PVK) because it acts as specific isolation medium for PSB isolation due to the presence of calcium triphosphate w hich is know n for 'halo' zone formation according to Sharma (2005). All the isolates w ere maintained at 4°C in equal volumes of nutrient broth and 30% glycerol.

Determ ination of soil physico-chem ical properties
The follow ing physico-chemical parameters of the soil w ere determined: pH, organic carbon, moisture content and phosphorus content. pH of the samples w as read using an electronic digital pH meter (Table 1). Moisture content w as determined by drying the samples in hot air oven at 105°C for 24 h using procedures outlined in the I.I.T.A. manual (IITA, 1979). The total organic carbon w as measured by the method given by according to APHA (1985). Available P w as determined by molybdenum blue method (Allen et al., 1974).

Biochem ical tests
All the isolates w ere characterized by Gram staining and biochemical tests follow ing the methods described by Olutiola et al. (1991). The various tests performed w ere oxidase, methyl red voges proskauer (MR-VP), indole, citrate, urease, H2S production and fermentation of various sugars. The morphological and biochemical characterization of the PSB isolates obtained from the rhizosphere of maize w as carried out w ith using 24 h old bacterial cultures. The results w ere compared w ith Bergey's Manual of Systematic Bacteriology (Holt et al., 1994).

In-vitro screening of isolates for phosphate solubilization activity
The isolates w ere screened for phosphate solubilization ability as described by Gupta et al. (1994). On modified Pikovskaya agar w ith insoluble tricalcium phosphate, a loop full of each culture w as placed on the center of agar plates and incubated at 30°C for 5 days. The solubilization zone w as determined by subtracting the diameter of bacterial colony from the diameter of total zone.

Distribution of PSB Isolates
A total of 23 isolates were obtained from the soil samples during the 56 days of sampling as shown in Table 2. Twenty-three phosphate solubilizing bacterial isolates designated as PSB were isolated from the different treatments of organic manure (OM), inorganic fertilizer (IF) and non-amended soil which served as control (CON) using Pikovskaya medium. The distribution of these isolates is presented in Table 2. Those isolates considered as PSB were those that formed a 'halo' on Pikovskaya medium. These isolates were subjected to different phosphate solubilization activity screening.

Total heterotrophic bacteria (THB) counts
The total heterotrophic bacteria (THB) within the rhizosphere of maize plants was counted using Nutrient Agar by pour plate method. THB counts in the rhizosphere of amended soil (organic manure and inorganic fertilizer) and non-amended control soil are presented in Figure 1. The total heterotrophic bacteria population in rhizosphere soil amended with organic  fertilizer increased from 15.4×10 9 CFU/g at day 14 to 26.8×10 9 CFU/g at day 56; that amended with inorganic fertilizer increased from 16.7×10 9 CFU/g to 25.2×10 9 CFU/g while that without amendment (Control) increased from 13.5×10 9 CFU/g to 20.1×10 9 CFU/g from day 14 to day 56, respectively. All through the period of study, the sample with organic manure treatment (OM) had the highest number of bacteria with 26.8×10 9 CFU/g followed by the inorganic fertilizer treatment (IF) having 25.2×10 9 CFU/g both at day 56. The inorganic fertilizer treatment had the highest bacterial count of 16.7×10 9 CFU/g at day 14. All the additives increased the microbial counts (population) during the period of the study more significantly compared to the control that had a lower microbial population.

Enumeration of PSB
Phosphate solubilizing bacteria (PSB) within the rhizosphere of maize plant was counted using Pikovskaya medium. The bacteria count of PSB isolates during the period of study is presented in Figure 2. The phosphate solubilizing bacteria in rhizosphere soil amended with organic manure (OM) increased from 6.3×10 9 CFU/g to 10.2×10 9 CFU/g; that amended with inorganic fertilizer (IF) increased from 6.0×10 9 CFU/g to 9.8×10 9 CFU/g while that without amendment (Control) increased from 6.1×10 9 CFU/g to 7.0×10 9 CFU/g from day 14 to day 56, respectively. The organic manure treatment had the highest bacterial count with 10.2×10 9 CFU/g followed by inorganic fertilizer treatment with 10.2×10 9 CFU/g at day 56. Here too, the amended soil treatments had higher growth potential compared to the control soil treatment.

Identification of PSB isolates
The results of the morphological and biochemical tests are presented in Table 3. The isolates belong to Pseudomonas sp. (13), Bacillus sp. (6) and Micrococcus sp. (2).

Screening of isolates for PSB activity
A total of twenty three (23) PSB isolates were screened for phosphate solubilization on modified Pikovskaya agar, of which eighteen (18) isolates showed the development of phosphate solubilization zones, ranging from 1.00 to 16.00 mm. Of the 18 isolates that showed zones of solubilization, 6 isolates (OMPSB5, CONPSB4, OMPSB8, IFPSB6, CONPSB5 had CONPSB6) had zones ranging between 6.00 and 16.00 mm while the other 12 isolates showed the development of zones less than 6.00 mm. OMPSB8 and IFPSB6 showed highest phosphate solubilization, that is, 16.00 and 15.00 mm, respectively. The zones of phosphate solubilization of PSB isolates are presented in Figure 3. Table 4 presents the highest solubilization zones of the isolates from the different treatments during each sampling period (day 14, 28, 42 and 56). For day 14, OMPSB2 had the highest solubilization of 3.00 mm. At day 28, the highest solubilization zone was by OMPSB3 with 5.00 mm. OMPSB5 and CONPSB4 both had highest solubilization zones of 6.00 mm at day 42. However, OMPSB8 and IFPSB6 showed highest phosphate solubilization zone, that is, 16.00 and 15.00 mm respectively at day 56.
Plant growth-promoting rhizobacteria (PGPR) colonize roots of plant and promote plant growth and development through a variety of mechanisms. The exact mechanism by which PGPR stimulate plant growth is not clearly known, although several mechanisms such as production  of phytohormones, suppression of deleterious organisms, activation of phosphate solubilization and promotion of the mineral nutrient uptake are usually believed to be involved in plant growth promotion (Bloemberg and Lugtenberg, 2001).
In the present study, 23 beneficial bacteria were isolated from maize rhizosphere. The bacteria isolates were screened for phosphate solubilization activities and characterized by biochemical tests. Generally, the total heterotrophic counts increased with increase in the duration of maize cultivation just as observed by Liang et al. (2012) in their 15 years study on the effect of fertilizers on soil quality. This can be attributed to the fact that the soil is a suitable medium for the growth of environmental microorganisms because it is rich in nutrients. It has also been reported that the soil receiving manure has larger bacteria pool than in the same soil receiving only chemical fertilizers (Islam and Weil, 2002).
The treatments with organic and inorganic amendments recorded higher bacteria counts as compared to the control treatment. This is similar to the report of Zhao et al. (2014). This observation may be as a result of the additional nutrients (N, P, K and micronutrients) provided by the additives which the bacteria breakdown for plant to use. This breakdown product in turn leads to the release of more exudates and plant products for use by the rhizosphere bacteria. Hence, increase in rhizosphere bacterial biomass (Das and Dkhar, 2011).
The addition of organic amendments increased the soil bacteria count compared to the inorganic fertilizer and control. Similar observations were made in organic recycling experiments by Chakrabarti et al. (2000) where soil receiving more organic matter tends to harbor higher levels of bacteria with higher microbial activity as proposed by Mäder et al. (2002). The higher bacteria count in OM treated soils is related to the organic matter with respect to decomposition in these materials which is important for proliferation of soil microorganisms in soil. The bacteria count for the organic manure treatments increased gradually over time and became higher than the inorganic fertilizer application which had a higher bacterial count at day 14. The explanation to this phenomenon can be deduced from the statement of Adegbidi et al. (2003) that the release of nutrients from composts and processed organic manures are generally slower than nutrient release from inorganic fertilizer. Kumar et al. (2012) isolated and identified Pseudomonas spp. as the major PSB in their research. They also reported that Pseudomonas spp. are very efficient phosphate solubilizers because they dissolve the soil P through production of low molecular weight organic acids in addition to lowering the pH of rhizosphere. This corroborates the findings in this study where more than 50% of the PSB isolated were identified as Pseudomonas sp.
The PSB isolated from the rhizosphere soil with organic manure treatment OMPSB8 (Pseudomonas sp.) showed the highest phosphate solubilization zone (16.00 mm) in PVK agar just as a documented by Lazcano et al. (2013). The reports of Zhang et al. (2012) and Yang et al. (2016) have also shown the positive effect of organic manure on the soil microflora. Furthermore, Kohler et al. (2015) showed that organic manure significantly increased shoot biomass by 64%. These reports and the current study show that organic manure application supports/enhances microbial activity in the rhizosphere of plants.

Conclusion
The application of organic and inorganic treatments significantly affected the rhizosphere bacterial population. Application of fertilizers showed increased bacterial population compared to the control treatment and this is of great importance in nutrient availability in the soil.
Similarly, the plant growth promoting activities of the isolates were also enhanced especially in the treatments with organic manure. The use of organic amendments is efficient, environmentally, cost effective and can be used in place of the more expensive inorganic fertilizer as a Richard and Ogunjobi 2027 viable alternative in the enrichment of nutrient deficient soil. Thus, it can be concluded that the application of organic manure can improve the soil health by improving the bacterial population. This type of study is necessary as it advocates that use of organic fertilizer is an efficient approach to replace chemical fertilizers.