Pyrroloquinoline quinone biosynthesis gene pqqC, a novel molecular marker for studying the phylogeny and diversity of phosphate-solubilizing pseudomonads

Abstract

Many root-colonizing pseudomonads are able to promote plant growth by increasing phosphate availability in soil through solubilization of poorly soluble rock phosphates. The major mechanism of phosphate solubilization by pseudomonads is the secretion of gluconic acid, which requires the enzyme glucose dehydrogenase and its cofactor pyrroloquinoline quinone (PQQ). The main aim of this study was to evaluate whether a PQQ biosynthetic gene is suitable to study the phylogeny of phosphate-solubilizing pseudomonads. To this end, two new primers, which specifically amplify the pqqC gene of the Pseudomonas genus, were designed. pqqC fragments were amplified and sequenced from a Pseudomonas strain collection and from a natural wheat rhizosphere population using cultivation-dependent and cultivation-independent approaches. Phylogenetic trees based on pqqC sequences were compared to trees obtained with the two concatenated housekeeping genes rpoD and gyrB. For both pqqC and rpoD-gyrB, similar main phylogenetic clusters were found. However, in the pqqC but not in the rpoD-gyrB tree, the group of fluorescent pseudomonads producing the antifungal compounds 2,4-diacetylphloroglucinol and pyoluteorin was located outside the Pseudomonas fluorescens group. pqqC sequences from isolated pseudomonads were differently distributed among the identified phylogenetic groups than pqqC sequences derived from the cultivation-independent approach. Comparing pqqC phylogeny and phosphate solubilization activity, we identified one phylogenetic group with high solubilization activity. In summary, we demonstrate that the gene pqqC is a novel molecular marker that can be used complementary to housekeeping genes for studying the diversity and evolution of plant-beneficial pseudomonads.

Fig. 1.

Phylogenetic relationships among 36 Pseudomonas reference strains, including known biocontrol strains described in Table 1. The neighbor-joining (NJ) trees were inferred from concatenated sequences of the two housekeeping genes rpoD and gyrB (1,130 bp) (A) and from pqqC (507 bp) sequences (B). Only bootstrap values greater than 50% are shown. Scale bar = 0.02 substitutions per site. Thin-lined box, P. syringae group; dashed-lined box, P. chlororaphis group; and thick-lined box, subgroup 1d (containing strains described as P. protegens by Ramette et al. [32]). Capital letters following the names of DAPG-producing strains indicate the multilocus group defined by Frapolli et al. (8).

Fig. 2.

Phylogenetic relationships among Pseudomonas reference strains and pseudomonads isolated from the rhizosphere of field-grown wheat in this study. The neighbor-joining (NJ) trees were inferred from concatenated sequences of the two housekeeping genes rpoD and gyrB (1,130 bp) (A) and from pqqC sequences (507 bp) (B). Only bootstrap values greater than 50% are shown. Scale bar = 0.02 substitutions per site. Both trees include sequences of 36 reference strains and of 136 pseudomonads isolated from wheat roots. Furthermore, the pqqC tree (B) includes an additional 107 partial pqqC sequences of noncultivated bacteria (cloned sequences). Pseudomonas isolates are described as follows: the first and the second number indicate the number of isolates per OTU and the OTU number, respectively, while C designates cultivated bacteria. Noncultivated pseudomonads are described by NC preceded by a figure indicating the number of cloned sequences and followed by a figure in parentheses describing the number of different OTUs identified from these sequences. Cultivated and noncultivated bacteria were obtained from the same wheat roots sampled in a Swiss field. Capital letters following the names of reference strains indicate the multilocus group defined by Frapolli et al. (8). Font colors indicate phosphate solubilization classes on NBRIP medium (based on halo diameter) as described in Materials and Methods and correspond to very low (violet), low (dark blue), medium (green), or strong (red) activity or undetermined activity (black).