Assessment of genetic and functional diversity of phosphate solubilizing fluorescent pseudomonads isolated from rhizospheric soil

Abstract

Background: Phosphorus is an essential macronutrient for the growth of plants. However, in most soils a large portion of phosphorus becomes insoluble and therefore, unavailable to plants. Knowledge on biodiversity of phosphate-solubilizing fluorescent pseudomonads is essential to understand their ecological role and their utilization in sustainable agriculture.

Results: Of 443 fluorescent pseudomonad strains tested, 80 strains (18%) showed positive for the solubilization of tri-calcium phosphate (Ca3(PO4)2) by the formation of visible dissolution halos on Pikovskaya's agar. These phosphate solubilizing strains showed high variability in utilizing various carbon sources. Numerical taxonomy of the phosphate solubilizing strains based on their carbon source utilization profiles resulted into three major phenons at a 0.76 similarity coefficient level. Genotypic analyses of strains by BOX (bacterial repetitive BOX element)-polymerase chain reaction (PCR) resulted into three distinct genomic clusters and 26 distinct BOX profiles at a 80% similarity level. On the basis of phenotypic characterization and 16S rRNA gene phylogenetic analyses strains were identified as Pseudomonas aeruginosa, P. mosselii, P. monteilii, P. plecoglossicida, P. putida, P. fulva and P. fluorescens. These phosphate solubilizing strains also showed the production of plant growth promoting enzymes, hormones and exhibited antagonism against phytopathogenic fungi that attack on various crops. Gene specific primers have identified the putative antibiotic producing strains. These putative strains were grown in fermentation media and production of antibiotics was confirmed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC).

Conclusion: Present study revealed a high degree of functional and genetic diversity among the phosphate solubilizing fluorescent pseudomonad bacteria. Due to their innate potential of producing an array of plant growth promoting enzymes, hormones and antifungal metabolites these phosphate solubilizing strains are considered to play a vital role in plant growth promotion, disease suppression and subsequent enhancement of yield.

Figure 1

Phosphate solubilization on Pikovskaya's agar medium by strain FPB9.

Figure 2

Phenogram of 80 phosphate solubilizing fluorescent pseudomonads based on their carbon source utilization profiles. The clustering was done using sequential, agglomerative, hierarchical and nested (SAHN) method. The pairwise coefficient of similarity (Dice) was used for clustering with the UPGMA algorithm using NTSYSpc2.02a software. The phenogram resulted into 3 major phenon at 0.76 smilarity coefficient. The experiment was done with three replicates.

Figure 3

Cluster analyses of BOX-PCR fingerprints showing the genotypic diversity of phosphate solubilizing fluorescent pseudomonads. Dendrogram was obtained from the similarity coefficient (Dice) calculations and clustering was done using unweighted pair-grouping method based on arithmetic averages (UPGMA) algorithm using BIOGENE software v11.02. The dendrogram resulted into 3 major clusters and 26 distinct BOX profiles.

Figure 4

Phylogenetic analyses of phosphate solubilizing strains of fluorescent pseudomonads based on the nucleotide sequence of 16S rRNA. The multiple sequence alignment was done in CLUSTAL program. The pair-wise evolutionary distances were calculated using Kimura2-parameter model. The phylogenetic tree was constructed by neighbor-joining (NJ) method with 1000 replicates using bootstrap. A total of 7 reference fluorescent pseudomonad strains were used for the tree construction.