Identification and characterization of inorganic-phosphate-solubilizing bacteria from agricultural fields with a rapid isolation method

Phosphorus (P), a non-renewable macronutrient, plays an essential role in plants (Elser et al. 2007). Inorganic P is mined to produce chemical P fertilizers that are extensively applied to cropland (Elser and Bennett 2011; Penuelas et al. 2013). The majority of soluble inorganic P, however, is rapidly immobilized by soil fixation and becomes unavailable for plant uptake, leading to low P-use efficiency and potentially excess P (Kochian 2012). Soil P must thus be managed to minimize its loss and increase its use efficiency.

Microorganisms are actively involved in many biogeochemical processes, including the mineralization, solubilization and transformation of soil P (van der Heijden et al. 2008). Inorganic-phosphate-solubilizing bacteria (iPSBs) are particularly effective in releasing P from pools of inorganic P. iPSBs can also prevent the liberated P from being fixed again (Richardson et al. 2009; Richardson and Simpson 2011). Screening highly efficient iPSBs as soil inoculum is a useful method for improving plant growth and yield (Richardson et al. 2009). Many methods for screening iPSBs have been reported (Chen et al. 2006; Chung et al. 2005; Mehta and Nautiyal 2001; Nautiyal 1999), but isolating iPSBs on separate petri dishes can be time-consuming and labor-intensive.

The mechanism of inorganic-P microbial mobilization is generally associated with extrusion of low-molecular-weight organic acids (Goldstein 1995), which can competitively chelate the cations bound to P via hydroxyl and carboxyl groups and convert them into soluble forms (Jones and Oburger 2011; Richardson and Simpson 2011). The solubilization of inorganic P, however, is complex and depends on numerous factors such as soil properties, plant nutritional requirements and physiological and growth conditions. Studies of the factors affecting solubilization are thus still needed.

We developed a rapid method for screening iPSBs and evaluating their effectiveness at solubilizing inorganic P, with an emphasis on potential highly efficient iPSBs for agricultural use. The isolated iPSB strains were identified and characterized. The types of organic acids secreted by the iPSB strains with high P-solubilizing abilities and the relationships between the organic acids, pH and P solubilization were analyzed.

Current agricultural practices tend to use sustainable technology, including the use of biofertilizers with multiple functions, to achieve high crop yields, which requires high soluble-P concentrations. iPSBs thus play an essential role in releasing P from soil-fixation systems and in preserving enough free phosphate in the rhizosphere for plant uptake and growth. iPSB inoculation can promote plant growth (Kaur and Reddy 2015; Yu et al. 2012), so identifying highly efficient iPSB strains thus becomes important. Our rapid screening using 96-well microplates provided an efficient method for simultaneously isolating numerous iPSB strains. The method isolated 76 positive iPSB strains within 72 h from two soils with three replicates. Each well has limited space, so only a single bacterial strain may survive if the sample is sufficiently diluted. Each well in our study contained a sole iPSB strain with a 10⁵ dilution of soil sample from two remote sites with different chemical properties, which was verified by incubation on 200-mm medium plates (Fig. 2), suggesting that further purification by a second or third colony transfer may not be needed. Positive iPSB strains were also easy to observe and count using bromocresol purple as an indicator.

Wells without bacterial growth and a color change were deemed to be negative, and yellow wells (definitely with bacterial growth) were deemed to be positive. Only the uncertain or ambiguous wells may need further confirmation. Two slightly yellow wells in our study were ultimately demonstrated to be positive iPSB strains with P-solubilizing ability. Our method can also effectively prevent the spread of fungal contamination. Most screening media contain glucose or other carbohydrates as carbon sources, so heterologous fungal spores can easily grow or be initially introduced in the diluted sample. Once a single fungal colony appears, it can promptly spread throughout the medium plate and interfere with screening. The first screening of iPSBs from soil samples also may need more than 72 h of incubation, so fungal survival is likely. Fungal spores in our method (Fig. 2a, purple circle), however, were confined to only one well instead of spreading and affecting surrounding wells. The rapid screening with 96-well microplates was demonstrated to be an efficient and effective way to isolate iPSB strains.

The identification of iPSB strains based on 16S rRNA sequences illustrated the phylogenetic structure of the potential iPSB communities in the two soils. B. megaterium contributed > 30% to the total iPSB population, which was also most frequently found in the Yingtan soil with an acidic pH (Fig. 3). Bacillus is abundant in various types of soil and has a strong P-solubilizing ability in cropland (Chen et al. 2006; Karagöz et al. 2012; Oliveira et al. 2009; Xuan et al. 2011). The next two most common genera, Arthrobacter and Streptomyces, which belong to Actinobacteria, are common in alkaline soils where considerable P is released (Aislabie et al. 2006; Gopalakrishnan et al. 2011; Xiong et al. 2012). Pseudomonas is a known iPSB genus and has been well studied due to genetic evidence of inorganic-P solubilization (Babu-Khan et al. 1995; Kwak et al. 2015; Umezawa et al. 2015). Other strains rarely act as iPSBs but can promote plant growth in various types of soils (Richardson et al. 2009; Rodrı́guez and Fraga 1999; Zhao et al. 2014).

In our study, B. megaterium was observed to solubilize more P than the other genera, including Streptomyces, Arthrobacter and Pseudomonas. The presence of B. megaterium in the rhizosphere could thus benefit plant P assimilation. B. megaterium isolates Y95, Y99, Y924 and Y1412 release > 130 μg mL⁻¹ soluble P when cultured in liquid PVK. Lactic and propionic acids were reported to be the two most common organic acids secreted by B. megaterium in a previous study (Chen et al. 2006), but we did not observe any ion-chromatographic peaks in the corresponding retention times of these two organic acids (Table 3). Gluconic acid has also been considered an important organic acid for P release (Rodríguez et al. 2006), but we did not detect this acid for any of the four representative iPSB strains. Of the three secreted organic acids, citric and oxalic acids are efficient P-solubilizing acids (Bolan et al. 1994), but their amounts released by these four strains were relatively low and may not be sufficient to liberate much P. The dominant succinic acid was not a common organic acid for P release, but the amounts secreted were strongly correlated with P release for the four representative strains over time (Fig. 4). Succinic acid concentration was strongly, positively and linearly correlated with P release (Fig. 5a) (R² = 0.7908, P < 0.001), suggesting that succinic acid was the main solubilizing acid secreted by B. megaterium.

Environmental pH and organic acid secretion were two common factors accounting for the mobilization of available P. pH is usually negatively correlated with the release of P (Chen et al. 2006; Rodrı́guez and Fraga 1999). The pH of the medium for some of the iPSBs in our study, however, was alkaline (Table 2), and the iPSBs likely had the ability to release P because they survived well at a high cell density (data not shown). The regression analysis of medium pH and P release (Fig. 5a) indicated a negative but not linear correlation. The pH may only sharply decrease with an increase in P release at acidic pHs, suggesting that soil pH may not be a universal factor to account for microbial P solubilization by iPSB stains.

We demonstrated the efficiency and effectiveness of this rapid iPSB screening method using 96-well microplates. This method is rapid and easy to manipulate and observe and can prevent the spread of fungal growth. B. megaterium was the main iPSB strain but released mostly succinic acid rather than other common organic acids for P solubilization. Our study may be useful for mechanistic study of microbial inorganic-P solubilization.