ns [37] . This is may be due to the difference in nitrogen source. Pikovskaya medium contains inorganic nitrogen (ammonium sulphate) while the nutrient agar contains organic nitrogen (beef extract and peptone) [36] . However, the red pigment, prodigiosin, results from secondary metabolism of Serratia marcescens [37] .

For more characterization of the recent isolates, resistance against some pesticides (captan, Thiophonate methyl, and oxamyl) is also tested and the results are recorded in Table 5. Generally, the isolates showed relatively high resistance against captan and Thiophonate methyl. Lower resistance was detected against oxamyl. However, both of Serratia marcescens strains scored the highest resistance levels against captan. On the other hand, Bacillus subtilis PH is the most resistant against thiophonate methyl. Resistance against pesticides, which are commonly used at the sampling area, is an advantage for phosphate solubilizing bacteria because it means more persistence in that harsh environment.

Finally, cell growth patterns and phosphate release of the three newly isolated strains as well as pH changes with time are clearly shown in Figures 1(a)-(d). Obviously, phosphate release which is in a direct proportion with OD475 and cell growth are increasing with days for all of the isolates. However, the mixed culture of the 3 isolates is the most efficient case in phosphate release (Figure 1(d)). On the other hand, pH is decreasing with time due to the secretion of organic acids into the medium for solubilization of calcium phosphate found in Pikovskaya broth [3] . The maximum drop in pH values was parallel with increased

Table 4. Morphological characterization of the new isolates.

Table 5. Resistance of the isolates against some commonly used pesticides.

Figure 1. Bacterial growth in Pikovskaya broth medium and phosphate release represented by the optical densities, OD, at 550 and 475, respectively, and culture pH values of the three strains ((a), (b), and (c)) and the mixed culture (d).

phosphate solubilization levels. The maximum acidification level was recorded for Serratia marcescens strain PH2 (pH = 1.94). However, in the mixed culture medium, pH dropped dramatically to 1 in 5 days from an initial point of 7. Phosphate solubilizing bacteria are usually produce lactic, gluconic, isobutyric, ketogluconic, oxalic, acetic, and citric acids. Besides, the mechanism of mineral phosphate solubilization is due to production of organic acids and/or phosphatases [38] [39] [40] . However, inorganic phosphate is solubilized by both organic and inorganic acids of phosphate solubilizing bacteria. Carboxyl and hydroxyl groups in these acids chelate Ca, Fe, and Al cations [41] . Usually, calcium phosphates (including rock phosphate ores) are insoluble in soil [41] . Gerretsen [42] reported that when pure cultures of soil bacteria are added to the soil, plant phosphate nutrition is increased throughout increased calcium-phosphate solubility. Soil pH is decreased in parallel and therefore phosphate solubilization is the net result of both pH decrease and acids production [43] . In other words, carboxylic and hydroxylic anions produced by phosphate solubilizing bacteria have high calcium affinity and therefore can solubilize more phosphorus than acidification alone [44] . Accordingly, there is a symbiotic relationship between plants and phosphate solubilizing bacteria [45] [46] , as bacteria provide the soluble phosphate and plant roots provide carbon compound such as sugars [47] . The net result of this relationship is crop production enhancement [48] [49] . The most significant solubilizers of phosphate are mainly belonging to Bacillus spp. such as B. subtilis, B. cereus, B. polymyxa, B. circulans, B. circalmous and B. megaterium [50] . Patil, 2014, [51] has reported that B. subtilis is a powerful phosphate solubilizer that tolerates soil salinity. Besides, phosphate solubilizing Bacillus megaterium mj1212 regulates endogenous plant carbohydrates and amino acids contents to promote Mustard plant growth [52] .

In 2008, Widiastuti [36] reported phosphate solubilization in Pikovskaya medium by Serratia marcescens and stated the relationship between phosphate solubilization and red pigment production. Previous studies reported the production of organic acids by Serratia marcescens [53] . Others proved the presence of genes such as pqq and gdh which are coding for phosphatase activity in Serratia marcescens [54] [55] as well as Pseudomonas [56] . Moreover, Lavania and Nautiyal [57] recorded that the soil isolate S. marcescens NBRI1213 is an efficient phosphate solubilizer and a potential plant growth promoting agent. Besides, Behera et al., and others [1] [58] [59] [60] [61] stated acid phosphatase production by Serratia. In addition to phosphate solubilization, Serratia and Alcaligenes faecalis have an antagonistic activity against plant pathogens [62] [63] [64] and can produce hydroxyl apatite [65] . In our research, the maximum phosphate solubilization efficiency was recorded in 5 days for the mixed culture followed by Serratia marcescens PH1. Previous studies recorded different periods to reach maximum phosphate solubilization. For instance, some researchers have reported 3, 4, 10, and even up to 15 days [66] .

4. Conclusions

In the present study, three different tomato rhizosphere bacterial strains are used for phosphate solubilization in Pikovskaya medium. These isolates are characterized by:

1) High phosphate solubilization index.

2) Increasing ability to release mineral phosphate over days with a dramatic decrease in pH values.

3) Resistance to pesticides that are commonly used in the sampling location.

All of these advantages make the bacterial isolates suitable as plant growth promoting symbionts that persist contamination conditions and make free phosphate anions available for plants.

Cite this paper
Mohamed, E. , Farag, A. and Youssef, S. (2018) Phosphate Solubilization by Bacillus subtilis and Serratia marcescens Isolated from Tomato Plant Rhizosphere. Journal of Environmental Protection, 9, 266-277. doi: 10.4236/jep.2018.93018.
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