Isolated Phosphate-Solubilizing Soil Bacteria Promotes In vitro Growth of Solanum tuberosum L.

The potato (Solanum tuberosum L.) is the third most important crop worldwide; its production demands a high supply of phosphorus (P) to promote the growth of roots and tubers (Dawwam et al. 2013). To supply these nutritional needs, 150 kg/ha of inorganic P is applied as a chemical fertilizer. However, inorganic P is accumulated in the soil by adsorption processes of metallic cations, such as Ca²⁺, Mg²⁺, Fe³⁺, and Al³⁺, becoming unavailable to the plant (Sharma et al. 2013). Consequently, soil fertility and its edaphic microbiota’s capacity to carry out biogeochemical cycles are decreasing (Dawwam et al. 2013; Sivasakthi et al. 2014).

Plant roots uptake inorganic P is as orthophosphate through non-symbiotic physiological mechanisms; however, through symbiosis with “plant-growth promoting Rhizobacteria” (PGPR), it facilitates, even more, their absorption (Richardson 2001; Richardson et al. 2009). PGPR solubilize P precipitated in the soil by synthesis and secretion of organic acids (malonic, gluconic, acetic, and lactic) either in aerobic or anaerobic metabolic pathways. Then, plant roots absorb the bioavailable orthophosphates (Sashidhar and Podile 2010). PGPR are also producers of plant hormone precursor metabolites and compounds with antagonistic activity to phytopathogens (Wani et al. 2007; Zaidi and Khan 2007; Ahmad et al. 2008).

Nowadays, the isolation of P-solubilizing PGPR is an issue of major interest because of their potential use as biofertilizers, which could reduce the use of agrochemicals that pollute and modify the structure and microbial community of soils (Ingle and Padole 2017). The P solubilization by isolated PGPR strains is evaluated on Pikovskaya agar plates using phosphate tricalcium as the only non-available phosphorus source. After an incubation period, bacterial colonies produce clear halos indicating P solubilization, and their diameter can be measured. Next, the bacterial strains are cultured in a liquid medium to quantify the released orthophosphates (Dawwam et al. 2013; Prathap and Ranjitha 2015).

Having identified the bacterial strain with the high phosphate solubilization (PSB), we hypothesize that the addition of PSB promotes potato plant-growth when cultured under in vitro conditions. This last part has not yet been studied, which is considered a critical methodological step before applying it in the field conditions to ensure proper implementation, as reported by Trdan et al. (2019).

The objectives of this study were: a) screening of PSB strains, isolated from agricultural soils of potato crops from central Mexico, and b) evaluation of the promotion of the plant growth with the use of a selected PSB strain in the in vitro potato cultures.

Screening and isolation of phosphate-solubilizing bacteria. Bacterial population density from soil samples was quantified to be 10⁵ CFU/g. Four out of total bacterial colonies present on nutrient agar were isolated for the subsequent growth on PVK agar and formation of the phosphate-solubilization halos. Fig. 1 shows that after eight days of incubation, the bacterial strain named A3 formed a 20 mm solubilization halo. Meanwhile, the other bacterial strains reached a solubilization halo of around 10 mm. However, it was necessary to confirm the P solubilization of strain A3 in a liquid medium to quantify the release of the orthophosphates.

Fig. 1.

Kinetics of growth and phosphate solubilization in PVK liquid medium. All four PSB strains presented a typical growth curve; however, exponential growth was more evident after three incubation days for bacterial strains A2 and A3. Although all bacterial strains were inoculated at the same optical density, bacterial strains A1 and A4 were less efficient to grow with tricalcium phosphate added under these experimental conditions (Fig. 2a).

Fig. 2.

On day three, during the exponential growth phase, both bacterial strains A2 and A3 solubilized 266 and 350 mg/l orthophosphates (Fig. 2b). The bacterial strains A1 and A4 solubilized 200 and 250 mg/l of orthophosphates, respectively. In this assay, all bacterial strains acidified the PVK liquid medium, but bacterial strains A2 and A3, in particular, acidified the medium with the highest efficiency as pH of the medium was equal to 5 (Fig. 2c).

The orthophosphates solubilization rates in PVK liquid medium were 88 mg P/l * d and 115 mg P/l * d for bacterial strains A2 and A3, respectively. Therefore, bacterial strain A3 was selected because it was able to grow with tricalcium phosphate being the only phosphorus source on both solid and liquid media, and it solubilized orthophosphates to a higher rate than bacterial strain A2.

The strain identification based on the 16S rRNA gene sequence. The 16S rDNA sequence was analyzed using the BLASTn algorithm and showed that bacterial strain A3 had 98% similarity with Bacillus pumilus and Bacillus zhangzhouensis. Fig. 3 shows the phylogenetic tree, where bacterial strain A3 appears as Fo03 strain (accession number MN100586), related to B. pumilus and B. zhangzhouensis.

Fig. 3.

Evaluation of P source and sucrose content in the development of potato microplants. Nodal cuttings incubated without any source of phosphate did not develop and died (T2 and T3). The cuttings developed with a source of phosphate and sucrose, specifically in T4 and T5, showed an increase in growth. It was found that there was a relationship between P source and sucrose concentration (Table I). Additionally, it was revealed that in medium supplemented with Ca₃(PO₄)₂, the pH value increased to 7.8. It did not affect microplant growth; therefore, in subsequent experiments, the culture medium pH was adjusted to this value.

Evaluation of in vitro growth of potato microplants in the presence of strain A3. Potato microplants incubated with tricalcium phosphate (as P source) and strain A3 presented a significant increase in the stem length by 79.3%, the fresh weight (2.2 times), and the root length by 68% when compared to control microplants (Table II).

An additional response observed in potato microplants inoculated with strain A3 was the overgrowth of adventitious roots compared to non-inoculated plants (Fig. 4).

Fig. 4.

In this paper, four out of sixteen bacterial strains from potato crop soil were screened and isolated. These four bacterial strains formed halos as evidence of P solubilization in PVK agar with tricalcium phosphate as non-soluble P source. Strain A3 had the highest halo diameter (20 mm) (Fig. 1). According to Paul and Sinha (2017), the use of PVK agar as a medium for qualitative estimation of P solubilization is considered the first step for screening and isolating PSB. Their paper reported a 13 mm halo diameter with the P-solubilizing strain of Pseudomonas aeruginosa.

The four bacterial strains were also cultured in PVK liquid medium to confirm and ensure that bacterial strain A3 was the best P solubilizer. The results showed that the P-solubilizing rate of bacterial strain A3 (115 mg P/l * d) was higher than those of A1, A2, and A4 strains. The results reported by Paul and Sinha (2017) showed that the P-solubilizing rate of Pseudomonas aeruginosa was 54 mg P/l * d.

After their isolation from the natural environment, the PSB has to grow under in vitro conditions, keep their solubilizing activity, and not be stressed in axenic cultures (Collavino et al. 2010). In this report, strain A3 maintained a high P-solubilization rate in PVK liquid medium, confirming its P-solubilization capacity, and it also acidified the culture medium to a pH of 5. It is well known that medium acidification is due to production and excretion of organic acids (gluconic, citric, lactic, succinic, oxalic, fumaric, acetic, isobutyric, glycolic, 2-ketogluconic, aspartic, and malonic); it is the primary microbial mechanism for solubilization of inorganic P (Rodriguez and Fraga 1999). Acidification modifies precipitation/dissolution equilibrium of P, organic acids sequester the calcium, and then the P is solubilized and becomes bioavailable (Prathap and Ranjitha 2015; Ingle and Padole 2017).

Phylogenetic analysis of the 16S rRNA gene showed that strain A3 is related to B. pumilus and B. zhangzhouensis. Although the cladogram shows that it is tightly and genetically related to B. pumilus, it is important to highlight that B. pumilus has not been reported to have phosphate-solubilizing activity. It has recently been identified as a PGPR capable of synthesizing phytohormone precursors, the compounds with the activity against nematodes (Okazaki et al. 2020). The bacteria also possess a chitinase activity in association with the rice crop roots, enhance carbohydrate metabolism and phenylpropanoid biosynthesis (Liu et al. 2020), and alleviate drought stress (Xie et al. 2019).

The strain of B. pumilus isolated from the P-solubilizing potato crop rhizosphere is reported here. There are relatively few reports of PGPR P solubilizers belonging to the genus Bacillus sp., isolated from potato rhizosphere. Such ability was detected in Bacillus strains isolated from potato rhizosphere from Peruvian soils by Calvo et al. (2010). Hanif et al. (2015) reported that Bacillus subtillis solubilizes phosphate and increases the length and weight of both roots and shoot in potatoes grown in the soils with the low phosphorus content.

Potatoes (S. tuberosum L.) need high doses of P for optimum growth and yield. When P is unavailable, growth and yield biomass are considerably reduced (Balemi 2009; Wang et al. 2015). The PSB helps plants to uptake P unavailable in soil, improving crop yield (Chen et al. 2006).

In this work, potato microplants were cultured in vitro with tricalcium phosphate and the PSB strain A3, to verify the bacterial effect on the promotion of plant growth. It is a frequent biotechnology procedure in tuber seed production systems (Ibrahim et al. 2016). It was found here that PSB could grow in both 10 or 30 g/l of sucrose (data not shown), and that there was a relationship between sucrose and source of phosphate in the development of potato microplants (Table I). It was observed that microplants significantly increased their development in Ca₃(PO₄)₂ that is not available to plants; this response could be attributed to the pH of the medium being adjusted to 5.6, which could contribute to the absorption of phosphorus by the plant.

A significant increase in stems and fresh weight was observed in potato microplants incubated with PSB strain A3 (Table II); it indicated that tricalcium phosphate was solubilized, and phosphate becomes available for plant growth. The increase in biomass could be attributed to phosphorus released by organic acids produced by PSB (Prieto-Correal et al. 2015). It has been shown that there is a correlation between the solubility of phosphorus and the production of organic acids by PSB (Chen et al. 2006). It has also been reported that, with low phosphate content available, plants can produce organic acids for phosphorus absorption (Wang et al. 2015). However, potato plants only produce succinic acid (Dechassa and Schenk 2004) in low concentration when compared to other crops (Wang et al. 2015). It suggests that phosphorus was made available due to the activity of the phosphate-solubilizing strain A3 and could be absorbed by potato microplants.

Besides, roots in potato microplants inoculated with phosphate-solubilizing strain A3 presented a statistically shorter length than those that were not inoculated (Table II). Various effects on roots have been reported according to the available P content. Ma and coworkers (Ma et al. 2001) found that roots of Arabidopsis thaliana were independent of the available P content, while in potato microplants, the number of roots increased when nutrients in the culture medium decreased (Ibrahim et al. 2016). In this work, potato microplants did not develop roots, possibly due to P bioavailability due to the effect of the phosphate-solubilizing strain A3.

In contrast, adventitious roots might increase significantly at low concentrations of available P, as demonstrated in Arabidopsis thaliana (Ma et al. 2001), potato, barley, and canola (Wang et al. 2015). A contrary response was found in the model used here with potato microplants inoculated in vitro with strain A3, where adventitious roots were formed, including in the main stem (Fig. 4). According to Ma and coworkers (Ma et al. 2001) P bioavailability alters root anatomy, allowing hairy roots formation for P acquisition.

It suggests that the potato microplant in the presence of tricalcium phosphate and phosphate-solubilizing strain A3 could have caused either: a) a vitrification phenomenon, which is a common tissue culture disorder due to excess of P available (Ziv 1993); it might lead to an increase in microplant weight due to thickening, but this response depended directly on the crop’s characteristics (Casas and Lasa 1986); or b) indole acetic acid production, which is characteristic of PGPR (Banerjee et al. 2010). It could induce lateral or adventitious roots, since a high auxin/cytokinin ratio might result in root formation (Rout 2004).

This investigation’s highlights are: phosphate-solubilizing bacteria occur in the soil, but this environment has high selection pressure; it was necessary to isolate and screen bacterial strains that show phosphate-solubilization activity. It was possible to select one out of four bacterial strains by culturing them on solid and in liquid medium with tricalcium phosphate. After molecular identification by sequencing of 16 S rRNA gene, it was demonstrated that the non-phytopathogenic strain A3 belongs to B. pumilus, being the first strain reported as a potato growth promoter. Finally, the in vitro assay with potato microplants showed that the supplementation with strain A3 reported herein promoted root and stem growth. This result may lead to future research on tuber formation in potato crops, and the bacteria may be used as biofertilizer in field conditions.