Prevalence and Correlates of Phenazine Resistance in Culturable Bacteria from a Dryland Wheat Field

Abstract

Phenazines are a class of bacterially produced redox-active natural antibiotics that have demonstrated potential as a sustainable alternative to traditional pesticides for the biocontrol of fungal crop diseases. However, the prevalence of bacterial resistance to agriculturally relevant phenazines is poorly understood, limiting both the understanding of how these molecules might shape rhizosphere bacterial communities and the ability to perform a risk assessment for off-target effects. Here, we describe profiles of susceptibility to the antifungal agent phenazine-1-carboxylic acid (PCA) across more than 100 bacterial strains isolated from a wheat field where PCA producers are indigenous and abundant. We found that Gram-positive bacteria are typically more sensitive to PCA than Gram-negative bacteria, and there was significant variability in susceptibility both within and across phyla. Phenazine-resistant strains were more likely to be isolated from the wheat rhizosphere, where PCA producers were also more abundant, compared to bulk soil. Furthermore, PCA toxicity was pH-dependent for most susceptible strains and broadly correlated with PCA reduction rates, suggesting that uptake and redox-cycling were important determinants of phenazine toxicity. Our results shed light on which classes of bacteria are most likely to be susceptible to phenazine toxicity in acidic or neutral soils. In addition, the taxonomic and phenotypic diversity of our strain collection represents a valuable resource for future studies on the role of natural antibiotics in shaping wheat rhizosphere communities. IMPORTANCE Microbial communities contribute to crop health in important ways. For example, phenazine metabolites are a class of redox-active molecules made by diverse soil bacteria that underpin the biocontrol of diseases of wheat and other crops. Their physiological functions are nuanced. In some contexts, they are toxic. In others, they are beneficial. While much is known about phenazine production and the effect of phenazines on producing strains, our ability to predict how phenazines might shape the composition of environmental microbial communities is poorly constrained. In addition, phenazine prevalence in the rhizosphere has been predicted to increase in arid soils as the climate changes, providing an impetus for further study. As a step toward gaining a predictive understanding of phenazine-linked microbial ecology, we document the effects of phenazines on diverse bacteria that were coisolated from a wheat rhizosphere and identify conditions and phenotypes that correlate with how a strain will respond to phenazines.

Keywords: correlates; dryland wheat; efflux pumps; phenazines; resistance; rhizosphere; toxicity.

FIG 1

Taxonomic distribution of bacterial isolates from wheat rhizosphere and bulk soil samples. This plot depicts the proportion of isolates from each sample that belonged to the 4 represented phyla. Each column represents one soil sample, and each box within the columns represents an individual isolate colored by the phylum to which it belongs (e.g., 6 boxes comprising one column indicate that 6 strains were isolated from that sample). B = Between (bulk soil), V = Virgin (bulk soil), W = Wheat (rhizosphere).

FIG 2

Distribution of PCA resistance phenotypes across phyla and soil sample type. (A and B) Heat maps depicting the growth of the strains at pH 5.1 (A) and pH 7.3 (B). Each row represents a strain. The left columns are colored according to the ratio of growth on PCA-containing agar versus PCA-free agar. Magenta indicates sensitivity to PCA while green indicates resistance to PCA. The right two columns in each heat map are colored according to the separate values for growth on PCA-containing agar (+PCA) or solvent control agar (−PCA) with darker green indicating more growth. Values are the mean of two to four biological replicates that each comprised three technical replicates. Asterisks indicate strains that displayed markedly variable susceptibility to PCA across biological replicates. Strains for which fewer than two biological replicates grew to stationary phase in the PCA-free control at the given pH, or for which color interfered with growth quantification, were omitted from this analysis. See Materials and Methods for a description of how growth was quantified. (C) Relative abundance of putative phenazine producers (phz+ bacteria) across the three types of soil samples. Error bars represent the standard deviation. (D and E) Density plots (i.e., smoothed histograms) representing the distribution of PCA resistance phenotypes at pH 5.1 (D) and pH 7.3 (E) among strains isolated from Between (bulk), Virgin (bulk), or Wheat (rhizosphere) soil. Higher values along the x-axis indicate greater resistance to PCA. If multiple identical strains were isolated from the same soil type, only one representative was counted. Thus, 26 strains were isolated from Between, 39 from Virgin, and 45 from Wheat. Colored tick marks above the x-axis represent where individual isolates fall in the range of PCA resistance phenotypes.

FIG 3

Growth of representative strains over time with and without PCA. Growth was quantified as described in the Methods. Solid lines represent the growth of spotted cultures on PCA-free agar, and dashed lines represent the growth of spotted cultures on agar containing 100 μM PCA. Blue represents growth at pH 7.3 and pink represents growth at pH 5.1. Data points are the mean of three technical replicates from a representative biological replicate for each strain, and the shaded ribbon represents the standard deviation. The selected strains depicted here cover the range of PCA susceptibility phenotypes observed in each genus. The full set of biological replicates for all tested isolates is in Fig. S4 to S7.

FIG 4

PCA reduction rates of selected strains at pH 5.1 and pH 7.3. (A) PCA reduction rates of representative strains from each phylum at pH 5.1 and pH 7.3. The values represent the mean of three biological replicates and error bars represent the standard deviation. Reduction rates were normalized to OD₆₀₀ values. (B) PCA reduction rates versus each strain’s growth ratio (+PCA/−PCA) at early stationary phase. The growth ratios are the same values calculated and used in Fig. 2.

FIG 5

Effects of efflux pump inhibitors on PCA susceptibility. (A and B) Growth curves of strains for which treatment with a combination of the efflux pump inhibitors (EPI) reserpine and PAβN did not affect susceptibility to PCA at pH 5.1 (A) or pH 7.3 (B). (C) Growth curves of strains for which treatment with reserpine and PAβN potentiated the toxicity of PCA. (D) Growth curves of strains for which treatment with reserpine and PAβN improved growth in the presence of PCA (strain W4I17) or vice versa (strains W1I16 and W4I9-1). In all panels, data points are the mean of three biological replicates and the shaded ribbon represents the standard deviation. The concentrations of reserpine and PAβN used for each strain can be found in Table S2.