2012 Annual Report
1a.Objectives (from AD-416):
To determine (1) the impact of water availability on the structure of the microbial community associated with phenazine production in the wheat rhizosphere; (2) the molecular mechanisms involved in survival of Phz+ bacteria in the rhizosphere of dryland wheat; and (3) how wheat health is affected by presence of elevated levels of PCA in the rhizosphere.
1b.Approach (from AD-416):
(1) The working hypothesis is that decreased soil moisture strongly affects the microbial rhizosphere community, leading to enrichment in Phz+ bacteria accompanied by increased production of PCA. Relationships between water potential, microbial community composition, and phenazine biosynthesis and turnover will be evaluated in greenhouse and field studies using pyrosequencing and qPCR with primers targeting 16S rDNA, phenazine and quorum-sensing (QS) genes, as well as reporter strains and HPLC-mass spectrometry. (2) Our working hypothesis is that Phz+ bacteria are uniquely adapted to colonize the plant rhizosphere under conditions of water stress due to their ability to resist desiccation via formation of robust biofilms. A draft genomic sequence of a Phz+ reference strain will be generated by pyrosequencing and used (i) to study the involvement of selected gene systems (QS circuits, polysaccharides, cyclic lipopeptides) in biofilm formation and resistance to desiccation; and (ii) to construct reporter strains for the analysis of gene expression in vitro and in situ in relation to soil moisture, root exudate constituents, and cross-communicating members of the rhizosphere microbial community. (3) The working hypothesis is that PCA affects plant gene expression and contributes to mineral reductive processes that act on rhizosphere Fe and Mn as a function of soil moisture. The AffymetrixTM Gene Chip will be used to assess the impact of PCA or Phz+ strains and isogenic Phz- mutants on wheat root gene expression under normal and water-stressed conditions. PCA and bacteria will also be evaluated for reductive dissolution of Fe and Mn in bulk soils and in the rhizosphere of seedlings maintained under dryland or irrigated conditions by standard colorimetric assays or by ICP-MS. Similar methods will be used to assess Fe concentrations in the tissues of field- and greenhouse-grown plants treated by Phz+ and Phz- bacteria.
Potential impact and expected outcomes: The proposed studies will provide insight into the impact of soil moisture on the rhizosphere microbial community structure of an economically important crop and the capacity of this community to produce a biologically active metabolite.
This research relates directly to objective 2a and b of the parent project because it describes the microbial communities in dryland wheat and the role of phenazines in bacterial survival. Root diseases, including take-all, Pythium, Rhizoctonia and common root rots, and Fusarium crown rot, cause $3.5 billion in losses annually to U.S. wheat and barley growers. For most of these diseases, there are no resistant varieties and chemical treatments are not available or are effective only during the seedling phase of the disease. Modern production practices of direct seeding, used to control soil erosion, exacerbates the incidence and severity of root diseases. This research focuses on fundamental microbe-plant interactions on the roots of wheat in order to develop effective biocontrol strategies that perform consistently against root diseases in sustainable cereal-based cropping systems. Specifically the research focuses on indigenous Pseudomonas bacteria that produce the antibiotic phenazine-1-carboxylate (PCA) on the roots of cereals grown in dryland agroecosystems, and on Pseudomonas strain 2-79, a well-studied biocontrol agent that produces PCA and was isolated from the roots of dryland wheat. The goal is to understand the factors leading to the enrichment of natural PCA-producing bacteria and their role in biocontrol and plant growth promotion in dryland wheat. To identify bacterial factors that contribute to the ability of PCA-producing bacteria to survive in dryland agroecosystems, ARS scientists at Pullman, Washington, in collaboration with researchers at Washington State University and Macquarie University, have determined and are annotating the DNA sequence of strain 2-79. To test the hypothesis that rhizosphere community structure and PCA production and activity are strongly influenced by soil moisture, paired dryland and irrigated wheat plots were established at the WSU Dryland Research Station at Lind, Washingotn, and this field study is in its second year. Roots of wheat grown with or without irrigation are being analyzed for the populations of PCA-producing bacteria, the amount of PCA produced on roots, and the total microbial communities associated with dryland and irrigated wheat roots. In the dryland wheat plot, the population size of PCA-producing bacteria on the roots remained high, whereas in the irrigated wheat, PCA-producing bacteria decreased throughout the growing season. Total rhizosphere DNA has been extracted and primers have been developed for community analysis of microbial populations in the rhizosphere of dryland and irrigated wheat. This two-year study is providing wheat growers with new approaches to make use of the natural biocontrol provided by PCA-producing bacteria. Accomplishments align with Component 3, Problem Statement 3B of NP 303.