1a. Objectives (from AD-416)
Identify factors, such as soil properties and plant species, that influence microbial community structure and function in order to develop management systems that optimize ecosystem services and maximize sustainability.
1b. Approach (from AD-416)
A. Eight Maryland soils were chosen that varied in pH, texture, organic C, and land use. After collection, soils were sieved and maintained at field moisture levels at 4 °C. Soil water potential curves were determined. Soils were incubated in Mason jars under controlled conditions to systematically vary soil moisture, soil temperature, soil pH, soil texture, and amendments with organic amendments. Soil samples are analyzed for microbial community structure by terminal restriction fragment length polymorphism (TRFLP) and phospholipid fatty acid (PLFA) analysis. B. Using the same soils as in (A), a variety of crop and weed seeds will be planted in each soil in pots, using 1 plant species per pot in a full factorial of plant species x soil with 6 replicates. Crop plant species will include cucumber (Cucumis sativus) and wheat (Triticum aestivum), while weed species will include smooth pigweed (Amaranthus hybridus), and giant foxtail (Setaria faberi). Soil and rhizosphere samples will be analyzed by TRFLP, PLFA, and pyrosequencing. C. We have a collection of 20 air-dried soils, collected in Maryland, that vary in pH, soil texture, and land use. Cucumber seeds will be planted in each soil and seedlings grown to select soils that will support cucumber growth with minimal disease. A subset of 8 soils will be chosen that maximize variation in pH and texture. Cucumber seeds will be planted in pots filled with these soils with and without prior seed inoculation with the biocontrol agent Pseudomonas fluorescens Pf-5. Colony forming units (CFU) of an antibiotic (rifampicin) resistant mutant of Pf5 will be determined by dilution-plating onto selective media containing rifampicin. As the cucumber plants grow rhizosphere soil samples (six replicates per treatment) will be taken and used directly to determine the population density of Pf-5 and frozen at -20 °C for analysis of rhizosphere community structure by TRFLP, PLFA, and pyrosequencing. D. We are collaborating with ARS scientists in a variety of locations, including Beltsville, MD; Watkinsville, Georgia; Brookings, South Dakota; Morris, MN; Prosser, WA; and Urbana, IL. They are using rye and/or vetch cover crops as part of their management systems. Bulk soil samples (0-15 cm depth) from three to four replicate plots, with treatments including rye, vetch, and bare soil, are being sent to us twice annually ( spring and fall) and being stored at -20 °C. Soil microbial community structure will be analyzed by TRFLP for Eubacteria, Archaea, and fungi, while soil enzyme assays will be used to measure community function.
3. Progress Report
This is the first report for this project that follows up on project 1265-12000-038-00D which terminated on May 2, 2011. Analysis of samples was continued from a series of soil incubation experiments to determine how major soil physical and chemical factors influence microbial community structure (Sub-Objective 1a). These experiments were designed to test the effects of fundamental soil properties (water potential, temperature, pH, texture, and organic matter) on soil microbial communities. A DNA-based approach, terminal restriction fragment length profiling, was used, but found to reveal fewer differences between treatments than an analysis of microbial lipids from the same samples. A more complex DNA-based approach is currently being tested in the hopes of achieving a more detailed analysis. An additional set of soil incubations, using a different soil type (Oxisol) was also initiated. Under Sub-Objective 1b, to determine the influence of plant species on rhizosphere communities in various soils, a greenhouse experiment was conducted to test the effects of a biocontrol agent on the cucumber rhizosphere community in 9 different soils. Samples were taken at multiple time points for future analysis. We have received soil samples from different cover cropping systems in different locations across the United States to identify soil and management factors influencing microbial community structure and function (Sub-Objective 1d). Samples from Brookings, SD, have been analyzed, and indicate that cover cropping with oats increases the biomass of mycorrhizae in the soil. Samples from other locations will be analyzed in 2012.
1. In collaboration with scientistst at MIDI Inc., ARS scientists at Beltsville, MD, have developed a high-throughput method for phospholipid fatty acid (PLFA) analysis of soils. PLFA analysis is used widely to measure microbial biomass and community composition, but is somewhat slow, expensive, and difficult to run on large numbers of samples at one time. We have adapted the method to run 96 samples simultaneously, and have also created a faster gas chromatographic method for use with the analysis. This new method promises to substantially decrease the time and expense needed for the analysis.