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. We sampled soil from the long-term farming systems experiment at the Center for Environmental Farming Systems (CEFS) Research Site (Goldsboro, NC). The sampling occurred after most plots were planted to corn (fall 2005), providing a “homogenized” soil sample with respect to recent crop history, a distinct advantage for the objectives of this grant. Homogenized and pooled soil samples representative of the various farming systems were analyzed by multiple investigators for nutrient status, biological indicators (microbial communities, nematode communities etc) and various physical parameters. Subsamples were immediately put in long-term storage for subsequent microarray analysis. Collaborative relationships were commenced, a postdoc was hired, and training has begun in micro-array analysis of soil microbial communities. A model system was enhanced to specifically ask questions associating function to the microbial communities in soils, particularly their impact on nitrogen and carbon cycling and plant health (i.e. plant disease suppression). A second postdoc has been recruited to focus on research efforts 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

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).

In 2005, due to the presence of a federal noxious weed, most plots were planted to corn (an unplanned systems change). This became an opportunity for the sake of our SARE work to examine microbial communities. Functionally, all plots were “homogenized” by crop allowing us to sample immediately after the corn crop was completed (September 2005). A common crop should decrease the variability in the microbial communities due to cropping sequence and help focus on farming systems effects. Approximately 30 to 40 soil cores were taken at each sampling and then mixed to near homogenization in the field and divided among several investigators for the various analyses. The project has investigated the types of organisms and characteristics that we believe are the most promising as indicators of the effects of each system including but not limited to micro-arthropod community structure, total microbial biomass, culturable bacteria (e.g. florescent pseudomonads and other culturable communities) and the structure of free-living and plant parasitic nematode populations. We also collected soils for nutrient and chemical analysis. Subsamples of all soils were immediately frozen at -80 C and -20 C for the purposes of our microarray analysis (Objective1). The measurement of all these characteristics from the same soil sample of known location is uncommon. By coordinating soil sampling, we hope to gain information on the effects of treatment on the interaction among physical, chemical, and biological components and on the spatial and temporal variation of their interactions.

Dr, Shuijin Hu and Dr. Frank Louws spent time at the University of Oklahoma in the early part of 2006 to work out collaborative plans for the micro-array analysis. Originally, Dr. J.-Z. Zhou from the Oak Ridge National Laboratory, Oakridge TN was identified as a participant in the original project and he was designated to perform microarray work at the national lab working with a postdoc. After we secured the grant, Dr. Zhou was offered and accepted a position at the University of Oklahoma-Norman Campus. With this change, we developed a subcontract between the University of Oklahoma and North Carolina State University. Dr. Zhou has recently hired a postdoc (in consultation with us) and is providing the direct supervision to accomplish the work we proposed in the funded proposal. The postdoc is currently being trained, but the move from Oakridge to Oklahoma has offset our timeline. Never-the-less, the proposed work, objectives and identified collaborator will remain the same and a subcontract was arranged to accommodate the change in location of the micro-array work. The postdoc is highly qualified for this work with superior ecology and sustainable agriculture experience to ensure basic science advancements that keeps an eye on the practical outcome.

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.

During the interim, we also have advanced a model system that provides a mechanism to address fundamental questions about mechanisms of disease suppression with regard to microbial communities in soils (especially as impacted by farming systems). In our most recent research (Dr. Shuijin Hu and colleagues) we examined the relationship between soil microbial diversity, and the invasion of a soilborne pathogen, Pythium ultimum. A microbial diversity gradient was created through reciprocally introducing microbial communities from three soils with significantly different microbial activities and diversity. The functional and biochemical diversity was quantified by characterizing microbial substrate utilization on Biolog Ecoplates and by the phospholipid fatty acid composition of the whole microbial community. Results obtained indicated that invasion and population density of P. ultimum was predominantly determined by soil resource availability (mainly available C and N). Both soil microbial diversity and P. ultimum population density were positively correlated to microbially available C (P < 0.01), leading to a negative relationship between diversity and pathogen density (P < 0.01). Thus, N and C cycling play a key role in plant health management and disease suppression. We have advanced this model system and will soon have in place a full time postdoc to address this Objective.

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 will be able to accomplish the work plan set out in the proposal (objective 2) within the time frame of the grant.

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 after most plots were planted to corn (fall 2005), providing a “homogenized” soil sample with respect to recent crop history, a distinct advantage for the objectives of this grant. • Homogenized and pooled soil samples representative of the various farming systems were analyzed by multiple investigators for nutrient status, biological indicators (microbial communities, nematode communities etc) and various physical parameters. Subsamples were immediately put in long-term storage for subsequent microarray analysis. • Collaborative relationships were commenced, a postdoc was hired, and training has begun in micro-array analysis of soil microbial communities. • A model system was enhanced to specifically ask questions associating function to the microbial communities in soils, particularly their impact on nitrogen and carbon cycling and plant health (i.e. plant disease suppression). • A second postdoc has been recruited to focus on research efforts 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 and plant growth promotion effects). The specific objectives with regard to this proposal have recently been initiated 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: