James A. Parejko

Diversity and Evolution of the Phenazine Biosynthesis Pathway

ABSTRACT Phenazines are versatile secondary metabolites of bacterial origin that function in biological control of plant pathogens and contribute to the ecological fitness and pathogenicity of the producing strains. In this study, we employed a collection of 94 strains having various geographic, environmental, and clinical origins to study the distribution and evolution of phenazine genes in members of the genera Pseudomonas, Burkholderia, Pectobacterium, Brevibacterium, and Streptomyces. Our results confirmed the diversity of phenazine producers and revealed that most of them appear to be soil-dwelling and/or plant-associated species. Genome analyses and comparisons of phylogenies inferred from sequences of the key phenazine biosynthesis (phzF) and housekeeping (rrs, recA, rpoB, atpD, and gyrB) genes revealed that the evolution and dispersal of phenazine genes are driven by mechanisms ranging from conservation in Pseudomonas spp. to horizontal gene transfer in Burkholderia spp. and Pectobacterium spp. DNA extracted from cereal crop rhizospheres and screened for the presence of phzF contained sequences consistent with the presence of a diverse population of phenazine producers in commercial farm fields located in central Washington state, which provided the first evidence of United States soils enriched in indigenous phenazine-producing bacteria.

Accumulation of the Antibiotic Phenazine-1-Carboxylic Acid in the Rhizosphere of Dryland Cereals

ABSTRACT Natural antibiotics are thought to function in the defense, fitness, competitiveness, biocontrol activity, communication, and gene regulation of microorganisms. However, the scale and quantitative aspects of antibiotic production in natural settings are poorly understood. We addressed these fundamental questions by assessing the geographic distribution of indigenous phenazine-producing (Phz+) Pseudomonas spp. and the accumulation of the broad-spectrum antibiotic phenazine-1-carboxylic acid (PCA) in the rhizosphere of wheat grown in the low-precipitation zone (<350 mm) of the Columbia Plateau and in adjacent, higher-precipitation areas. Plants were collected from 61 commercial wheat fields located within an area of about 22,000 km2. Phz+ Pseudomonas spp. were detected in all sampled fields, with mean population sizes ranging from log 3.2 to log 7.1 g−1 (fresh weight) of roots. Linear regression analysis demonstrated a significant inverse relationship between annual precipitation and the proportion of plants colonized by Phz+ Pseudomonas spp. (r 2 = 0.36, P = 0.0001). PCA was detected at up to nanomolar concentrations in the rhizosphere of plants from 26 of 29 fields that were selected for antibiotic quantitation. There was a direct relationship between the amount of PCA extracted from the rhizosphere and the population density of Phz+ pseudomonads (r 2 = 0.46, P = 0.0006). This is the first demonstration of accumulation of significant quantities of a natural antibiotic across a terrestrial ecosystem. Our results strongly suggest that natural antibiotics can transiently accumulate in the plant rhizosphere in amounts sufficient not only for inter- and intraspecies signaling but also for the direct inhibition of sensitive organisms.

Recent insights into the diversity, frequency and ecological roles of phenazines in fluorescent Pseudomonas spp.

Phenazine compounds represent a large class of bacterial metabolites that are produced by some fluorescent Pseudomonas spp. and a few other bacterial genera. Phenazines were first noted in the scientific literature over 100 years ago, but for a long time were considered to be pigments of uncertain function. Following evidence that phenazines act as virulence factors in the opportunistic human and animal pathogen Pseudomonas aeruginosa and are actively involved in the suppression of plant pathogens, interest in these compounds has broadened to include investigations of their genetics, biosynthesis, activity as electron shuttles, and contribution to the ecology and physiology of the cells that produce them. This minireview highlights some recent and exciting insights into the diversity, frequency and ecological roles of phenazines produced by fluorescent Pseudomonas spp.

Biological Control of Take-All by Fluorescent Pseudomonas spp. from Chinese Wheat Fields

Take-all disease of wheat caused by the soilborne fungus Gaeumannomyces graminis var. tritici is one of the most important root diseases of wheat worldwide. Bacteria were isolated from winter wheat from irrigated and rainfed fields in Hebei and Jiangsu provinces in China, respectively. Samples from rhizosphere soil, roots, stems, and leaves were plated onto Kings medium B agar and 553 isolates were selected. On the basis of in vitro tests, 105 isolates (19% of the total) inhibited G. graminis var. tritici and all were identified as Pseudomonas spp. by amplified ribosomal DNA restriction analysis. Based on biocontrol assays, 13 strains were selected for further analysis. All of them aggressively colonized the rhizosphere of wheat and suppressed take-all. Of the 13 strains, 3 (HC9-07, HC13-07, and JC14-07, all stem endophytes) had genes for the biosynthesis of phenazine-1-carboxylic acid (PCA) but none had genes for the production of 2,4-diacetylphloroglucinol, pyoluteorin, or pyrrolnitrin. High-pressure liquid chromatography (HPLC) analysis of 2-day-old cultures confirmed that HC9-07, HC13-07, and JC14-07 produced PCA but no other phenazines were detected. HPLC quantitative time-of-flight 2 mass-spectrometry analysis of extracts from roots of spring wheat colonized by HC9-07, HC13-07, or Pseudomonas fluorescens 2-79 demonstrated that all three strains produced PCA in the rhizosphere. Loss of PCA production by strain HC9-07 resulted in a loss of biocontrol activity. Analysis of DNA sequences within the key phenazine biosynthesis gene phzF and of 16S rDNA indicated that strains HC9-07, HC13-07, and JC14-07 were similar to the well-described PCA producer P. fluorescens 2-79. This is the first report of 2-79-like bacteria being isolated from Asia.

Irrigation Differentially Impacts Populations of Indigenous Antibiotic-Producing Pseudomonas spp. in the Rhizosphere of Wheat

ABSTRACT This work determined the impact of irrigation on the seasonal dynamics of populations of Pseudomonas spp. producing the antibiotics phenazine-1-carboxylic acid (Phz+) and 2,4-diacetylphloroglucinol (Phl+) in the rhizosphere of wheat grown in the low-precipitation zone (150 to 300 mm annually) of the Columbia Plateau of the Inland Pacific Northwest. Population sizes and plant colonization frequencies of Phz+ and Phl+ Pseudomonas spp. were determined in winter and spring wheat collected during the growing seasons from 2008 to 2009 from selected commercial dryland and irrigated fields in central Washington State. Only Phz+ bacteria were detected on dryland winter wheat, with populations ranging from 4.8 to 6.3 log CFU g−1 of root and rhizosphere colonization frequencies of 67 to 100%. The ranges of population densities of Phl+ and Phz+ Pseudomonas spp. recovered from wheat grown under irrigation were similar, but 58 to 100% of root systems were colonized by Phl+ bacteria whereas only 8 to 50% of plants harbored Phz+ bacteria. In addition, Phz+ Pseudomonas spp. were abundant in the rhizosphere of native plant species growing in nonirrigated areas adjacent to the sampled dryland wheat fields. This is the first report that documents the impact of irrigation on indigenous populations of two closely related groups of antibiotic-producing pseudomonads that coinhabit the rhizosphere of an economically important cereal crop. These results demonstrate how crop management practices can influence indigenous populations of antibiotic-producing pseudomonads with the capacity to suppress soilborne diseases of wheat.

Taxonomy and Distribution of Phenazine-Producing Pseudomonas spp. in the Dryland Agroecosystem of the Inland Pacific Northwest, United States

ABSTRACT We investigated the taxonomic placement of phenazine-producing fluorescent Pseudomonas spp. in the Inland Pacific Northwest region of the United States. Five distinct species were identified, two of which were provisionally considered to be new. Agroclimatic zone and soil silt content affected the species diversity across the region.

Sensitivity of Rhizoctonia Isolates to Phenazine-1-Carboxylic Acid and Biological Control by Phenazine-Producing Pseudomonas spp.

Rhizoctonia solani anastomosis groups (AG)-8 and AG-2-1 and R. oryzae are ubiquitous in cereal-based cropping systems of the Columbia Plateau of the Inland Pacific Northwest and commonly infect wheat. AG-8 and R. oryzae, causal agents of Rhizoctonia root rot and bare patch, are most commonly found in fields in the low-precipitation zone, whereas R. solani AG-2-1 is much less virulent on wheat and is distributed in fields throughout the low-, intermediate-, and high-precipitation zones. Fluorescent Pseudomonas spp. that produce the antibiotic phenazine-1-carboxylic acid (PCA) also are abundant in the rhizosphere of crops grown in the low-precipitation zone but their broader geographic distribution and effect on populations of Rhizoctonia is unknown. To address these questions, we surveyed the distribution of PCA producers (Phz+) in 59 fields in cereal-based cropping systems throughout the Columbia Plateau. Phz+ Pseudomonas spp. were detected in 37 of 59 samples and comprised from 0 to 12.5% of the total culturable heterotrophic aerobic rhizosphere bacteria. The frequency with which individual plants were colonized by Phz+ pseudomonads ranged from 0 to 100%. High and moderate colonization frequencies of Phz+ pseudomonads were associated with roots from fields located in the driest areas whereas only moderate and low colonization frequencies were associated with crops where higher annual precipitation occurs. Thus, the geographic distribution of Phz+ pseudomonads overlaps closely with the distribution of R. solani AG-8 but not with that of R. oryzae or R. solani AG-2-1. Moreover, linear regression analysis demonstrated a highly significant inverse relationship between annual precipitation and the frequency of rhizospheres colonized by Phz+ pseudomonads. Phz+ pseudomonads representative of the four major indigenous species (P. aridus, P. cerealis, P. orientalis, and P. synxantha) suppressed Rhizoctonia root rot of wheat when applied as seed treatments. In vitro, mean 50% effective dose values for isolates of AG-8 and AG-2-1 from fields with high and low frequencies of phenazine producers did not differ significantly, nor was there a correlation between virulence of an isolate and sensitivity to PCA, resulting in rejection of the hypothesis that tolerance in Rhizoctonia spp. to PCA develops in nature upon exposure to Phz+ pseudomonads.

Long-Term Irrigation Affects the Dynamics and Activity of the Wheat Rhizosphere Microbiome

The Inland Pacific Northwest (IPNW) encompasses 1. 6 million cropland hectares and is a major wheat-producing area in the western United States. The climate throughout the region is semi-arid, making the availability of water a significant challenge for IPNW agriculture. Much attention has been given to uncovering the effects of water stress on the physiology of wheat and the dynamics of its soilborne diseases. In contrast, the impact of soil moisture on the establishment and activity of microbial communities in the rhizosphere of dryland wheat remains poorly understood. We addressed this gap by conducting a three-year field study involving wheat grown in adjacent irrigated and dryland (rainfed) plots established in Lind, Washington State. We used deep amplicon sequencing of the V4 region of the 16S rRNA to characterize the responses of the wheat rhizosphere microbiome to overhead irrigation. We also characterized the population dynamics and activity of indigenous Phz+ rhizobacteria that produce the antibiotic phenazine-1-carboxylic acid (PCA) and contribute to the natural suppression of soilborne pathogens of wheat. Results of the study revealed that irrigation affected the Phz+ rhizobacteria adversely, which was evident from the significantly reduced plant colonization frequency, population size and levels of PCA in the field. The observed differences between irrigated and dryland plots were reproducible and amplified over the course of the study, thus identifying soil moisture as a critical abiotic factor that influences the dynamics, and activity of indigenous Phz+ communities. The three seasons of irrigation had a slight effect on the overall diversity within the rhizosphere microbiome but led to significant differences in the relative abundances of specific OTUs. In particular, irrigation differentially affected multiple groups of Bacteroidetes and Proteobacteria, including taxa with known plant growth-promoting activity. Analysis of environmental variables revealed that the separation between irrigated and dryland treatments was due to changes in the water potential (Ψm) and pH. In contrast, the temporal changes in the composition of the rhizosphere microbiome correlated with temperature and precipitation. In summary, our long-term study provides insights into how the availability of water in a semi-arid agroecosystem shapes the belowground wheat microbiome.

The Role of 2,4-Diacetylphloroglucinol- and Phenazine-1-Carboxylic Acid-Producing Pseudomonas spp. in Natural Protection of Wheat from Soilborne Pathogens

Fluorescent Pseudomonas isolated from the rhizosphere of diverse plants have been studied as biocontrol agents of soilborne pathogens worldwide. Certain strains of these bacteria are capable of exerting a variety of mechanisms of plant growth promotion and protection, including the production of the natural antibiotics 2,4-diacetylphloroglucinol (2,4-DAPG) and derivatives of phenazine (Phz). These compounds are broadly active against plant pathogens and are produced by widely distributed taxonomically diverse Pseudomonas spp. that inhabit the rhizosphere of cereal crops and render certain soils naturally suppressive to soilborne plant diseases. There is mounting evidence that the role of 2,4-DAPG and phenazines in the rhizosphere is not limited to antibiosis but also involves regulatory and signaling functions, induction of systemic resistance, and reduction of minerals in soil.