Microarray Analysis and functional assays to assess microbial ecology and disease suppression in soils under organic or sustainable management

Microarray Analysis and functional assays to assess microbial ecology and disease suppression in soils under organic or sustainable management

Summary

The purpose of this project is to characterize soil microbial communities and understand (and ideally learn how to manage) the links between microbial community structure and diversity and ecological function. Fundamental ecological functions consistent with our expertise include nutrient cycling, plant disease suppression, and plant growth promoting effects as mediated by soil microbes and impacted by long-term farming systems. [Plant disease suppression refers to the combined abiotic and biotic components of the soil that limit the ability of plant pathogens to invade the soil, limit the persistence of plant pathogens in the soil, and/or modulate the host such that it is less susceptible i.e. mediated by induced or systemic acquired resistance. Plant growth promoting effects refer to the plant’s response to the soil environment and could be a general response to enhanced soil quality such as increased water holding capacity or availability of nutrients but, more specifically, it relates to the function of soil microbes associated with plant roots (e.g. rhizosphere, rhizoplane, or endosphere) that specifically enhance plant growth, possibly mediated by enhanced nutrient acquisition, plant growth hormone production, or reduced disease incidence, among other possible mechanisms]. Our interests focus primarily on the microbial component of these beneficial functions that we hypothesize can be enhanced in sustainable and organic farming systems. Collaborative relationships were commenced in 2006 (see 2005 report), a postdoc was hired in Oklahoma, and she has been trained in micro-array analysis of soil microbial communities. Sample delivery was delayed due to the need to secure a federal permit to transport the soil samples (due to the presence of an identified federally noxious weed in our long-term farming systems). Analysis of the microbial communities will look at large scale spatial diversity of soil microbes and the impact of diverse farming systems (baseline 1999 and terminal sampling of 2005) with particular emphasis on C & N dynamics. As a side-benefit, new microarrays have been developed with a greater diversity of genes. A second series of soil samples were collected in the fall of 2006 to look at the detailed spatial dynamics of soil microbes over relatively small distances (2.5 to 250 cm). Samples were collected from conventional tillage plots, no till plots (that had the same cropping sequence since 1999) and the successional plot (natural succession of species since 1999). These samples were analyzed by multiple investigators for nutrient status, biological indicators (microbial communities, nematode communities etc), respiration, microbial biomass N & C, net N mineralization and various physical parameters. Subsamples were immediately put in long-term storage for subsequent microarray analysis. Samples were recently delivered to Oklahoma. A second post-doc was recently hired to conduct molecular-based work on the diversity of specific soil populations and to elucidate mechanisms of disease suppression in long term SARE organic and farming systems projects. We foresee the practice and science of sustainable agriculture will advance through implementation of visionary systems-based practices (such as at CEFS) informed by fundamental knowledge obtained by component research such as linking soil microbial community diversity and dynamics to ecological functions that benefit production agriculture.

Objectives/Performance Targets

Objectives/Performance Targets:
OBJECTIVE 1: To utilize microarray technology to assess microbial diversity and structure as impacted by long-term farming systems structure with emphasis on microbial communities associated with nutrient cycling and disease suppression.

Our research is being accomplished within the framework of a large farming systems experiment at the Center for Environmental Farming Systems (CEFS) near Goldsboro, North Carolina. Five farming systems were implemented in 1999 and include varying levels of disturbance and carbon inputs. The CEFS site, established in 1998, is dedicated to interdisciplinary analyses focusing on systems approaches to pest/crop management with an emphasis on soil quality. A series of systems-based analyses have been conducted at the CEFS site under various treatment/rotation conditions. Approximately 81 ha (200+ acres) have been divided, based on intensive soil mapping, into three replications of five treatments. Individual subplots vary in size from about 1.2 to 3.8 ha, depending on the treatment and the replicate. Soils were intensively mapped in 1996 based on soil type and drainage. Replications were designated based on this mapping, with a similar ‘diagnostic’ soil type available for sampling in each treatment, characteristic of each individual replication. Five, permanent, geo-referenced sampling points have been designated in the diagnostic soil of each sub-plot in each treatment. Soil sampling has been coordinated between researchers allowing us to gain information of the effects of treatment on the interaction among physical, chemical, and biotic components and on the spatial and temporal variation of their interaction. Soil was sampled to a depth of 15 cm from the five GPS mapped locations (in the crop row) in each treatment. Samples have been at 3-4 time points from the early spring through the summer over the duration of the study (1999-2005).

Collaborative relationships were commenced in 2006 (see 2005 report), a postdoc was hired in Oklahoma, and she has been trained in micro-array analysis of soil microbial communities. Sample delivery was delayed due to the need to secure a federal permit to transport the soil samples (due to the presence of an identified federally noxious weed in our long-term farming systems). Analysis of the microbial communities will look at large scale spatial diversity of soil microbes and the impact of diverse farming systems (baseline 1999 and terminal sampling of 2005) with particular emphasis on C & N dynamics. As a side-benefit, new microarrays have been developed with a greater diversity of genes. A second series of soil samples were collected in the fall of 2006 to look at the detailed special dynamics of soil microbes over relatively small distances (2.5 to 250 cm). Samples were collected from conventional tillage plots, no till plots (that had the same cropping sequence since 1999) and the successional plot (natural succession of species since 1999). These samples were analyzed by multiple investigators for nutrient status, biological indicators (microbial communities, nematode communities etc), respiration, microbial biomass N & C, net N mineralization and various physical parameters. Subsamples were immediately put in long-term storage for subsequent microarray analysis. Samples were recently delivered to Oklahoma.

Dr. Frank Louws spent time at the University of Oklahoma in July of 2006 to interact with the Oklahoma collaborators and an Oklahoma posrtdoc cam e in October 2006 to sample for the small-scale spatial analysis.

OBJECTIVE 2: To elucidate mechanisms of disease suppression in long term SARE organic and farming systems projects.

Much research in microbial ecology tends to be descriptive, monitoring the diversity and dynamics of microbial communities or specific members of the community. A greater problem lies in associating function to the communities. We are interested in the function of microbial communities, particularly their impact on nitrogen and carbon cycling and plant health (i.e. plant disease suppression). From selected CEFS samples of 2005, we have performed microbial biomass C by fumigation-extraction and microbial biomass N following alkaline persulfate oxidation. The microbial community structure was also characterized by analyzing the phospholipid fatty acid (PLFA) biomarkers and a culture-based assessment of total (culturable) bacteria, fluorescent pseudomonad populations and other specific microbial populations. This data has not been compiled in its entirety to date.

In the original proposal we sought to support a graduate student and part time technical help. We were not able to secure a superior graduate student for this work and did not want to compromise the work with just any student. Therefore, we adjusted all the proposed salary monies (original budget less the University of Oklahoma sub-contract) and allocated the funds into a postdoc position, in full communication with the Southern Region SARE office and NCSU financial personnel. This provided enough money to hire a postdoc. This postdoc was hired 01-2007 after several delays and will be able to accomplish the work plan set out in the proposal (objective 2) within the time frame of the grant. To date, the post-doc has developed protocols to evaluate the genetic diversity and dynamics of specific microbial populations (particularly Burkholderia species) that have been isolated form the farming systems experiment from 1999-2005. Burkholderia populations are particularly interesting due to their diverse ecological functions including biological control of plant diseases and plant growth promoting effects.

Accomplishments/Milestones

Accomplishments/Milestones
• Soils were sampled from the long-term farming systems experiment at the Center for Environmental Farming Systems Research Site (Goldsboro, NC). The sampling occurred in three “farming systems” to look at the spatial dynamics of microbial communities with a limited area (2-250 cm).
• Homogenized and pooled soil samples representative of selected distances (2—250 cm) were analyzed by multiple investigators for nutrient status, biological indicators (microbial communities, nematode communities), respiration, microbial biomass N & C, net N mineralization and various physical parameters. Subsamples were immediately put in long-term storage for subsequent microarray analysis and delivered to Oklahoma for microarray analysis.
• A postdoc has been trained in micro-array analysis of soil microbial communities.
• A second postdoc has been hired to focus on research efforts to assess the genetic diversity and dynamics of selected microbial communities and to elucidate mechanisms of disease suppression in long term SARE organic and farming systems projects.

Impacts and Contributions/Outcomes

Our work is part of an ongoing interdisciplinary project. This interdisciplinary approach will afford a broad understanding of the individual soil microenvironments associated with each sampling site and farming systems plots. All experimental parameters are being collected and sorted into a merged internet-based master database to facilitate cross analyses of data from different investigators and to promote interdisciplinary studies. This database is an unprecedented resource to compare the results of our microarray analysis. It is anticipated the tools, protocols, and basic biology information we desire to develop will enable an enhanced understanding of how organic soils and whole-farm management systems (organic, sustainable) impact microbial diversity and structure and the impact these communities have on specific functions (e.g. disease suppression, plant growth promotion effects and nutrient cycling). The specific objectives with regard to this proposal are underway and direct impacts are anticipated to be forthcoming. We foresee the practice and science of sustainable agriculture will advance through implementation of visionary systems-based practices (such as at CEFS) informed by fundamental knowledge obtained by component research that addresses key problems, as articulated in our 2 objectives and current work.

Collaborators: