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

This project was highly productive. A few key points bear highlighting. First, we are engaged in sustainable agriculture research, teaching and extension at multiple levels – from the field to national and international programming. We support Southern SARE’s desire to conduct component research, farming systems research and more recently research that involves sociological questions and advancing agriculture within a sustainable society. Advances need to occur at all levels. This project focused on component research and sought to advance the fundamental understanding of the impact of farming systems on microbial communities and then to advance our knowledge about the link between microbial communities and plant disease incidence and crop productivity. We observed that the science of microbial ecology is exploding in terms of the ability of scientists to characterize whole communities and to analyze the links between communities and ecosystem services (e.g. plant disease suppression, N-Cycling; C-Cycling etc) through the emergence of new and complex statistical tools and bioinformatics software. We systematically sampled conventional tillage, no-tillage and successional ‘farming’ systems initiated in the spring of 1999 and assessed physical, chemical and biological soil properties. Soybean yields were also assessed at soil sampling in the fall of 2007. Culture-based methods were used to count the density of Burkholderia, Pythium and Fusarium species. DNA was also extracted from soils and analyzed for diversity of these same species – linking culture-based and molecular-based approaches to assessing microbial diversity of soils. DNA was also extracted and is currently being analyzed based on GeoChip Analysis using a novel comprehensive microarray design that has ~25,000 probes and covers ~47,000 sequences for 292 gene families involved in nitrogen, carbon, sulfur and phosphorus cycling, metal reduction and resistance, and organic contaminant degradation. The data shows microbial community diversity over space follows a different ecological law than the diversity of eukaryotes. Also, we have partitioned the diversity into variables associated with chemical, physical and spatial components of the fields. We anticipate that we will be able to take the biological, physical and chemical parameters and advance the understanding of the complex inter-relationships of plant pathogens, soil microbes, disease incidence and soybean yield using tools that employ random matrix theory applications and other types of statistical tools. Although this work is more fundamental, the project generated applied talks at conferences to enable growers to think about the role of microbial communities in soil. Likewise, the project enabled us to engage scientists at the national and international level – particularly on the importance of microbial ecology in sustainable agricultural systems. For example, Dr. Louws attended a series of international microbial ecology meetings in Europe to present data from this work and interact with scientists about driving questions, methods and outcomes of microbial ecology research in sustainable farming systems.

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. Collaborative relationships were commenced in 2006 (see 2005 & 2006 report), a postdoc was hired in Oklahoma to conduct the micro-array analysis of soil microbial communities. After working on the project for a short time, the postdoc felt this was the wrong area of work and elected to pursue a Law Degree. Another post-doc was hired and work recommenced. Another series of soil samples were collected in the fall of 2007 to look at the detailed spatial dynamics of soil microbes over defined distances (0.5 m to 500 m) and as impacted by long-term farming systems. Twenty-one samples per plot were collected from conventional tillage, no till (that had the same cropping sequence since 1999) and successional plots (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 and delivered to Oklahoma.

Many of the soil physical, chemical and biological factors have been assessed. Data analyzed to date are included here in preliminary format. Current work includes multivariate analysis to link relationships between physical, chemical and biological parameters.
1. Soil physical parameters in soils with long-term conventional tillage (CT), no-tillage (NT) and successional (SC) systems: Soil bulk density and soil porosity were significantly higher in soils with no-tillage and conventional tillage than those with successional systems. Soil water content was highest in soils with no-tillage, followed by conventional tillage and successional systems. Soil clay was significantly higher in soils with no-tillage and conventional tillage than those with successional systems. Soil silt was significantly higher in soils with no-tillage than those with conventional tillage or successional systems. Soil sand was significantly higher in soils with no-tillage and successional than those with conventional tillage systems.
2. Soil chemical parameters in soils with long-term tillage (CT), no-tillage (NT) and successional (SC) systems. Cation exchange capacity was similar in soils with conventional tillage, no-tillage and successional systems: pH was highest in soils with conventional tillage, followed by the soils with no tillage and successional systems with significant difference. Humic matter was higher in soils with no-tillage than those with conventional tillage and successional systems. Nitrogen release was higher in soils with no-tillage than those with conventional tillage and successional systems. Soluble Sulfur was highest in soils with successional systems, followed by conventional tillage and no tillage systems with significant difference. Calcium was higher in soils with conventional tillage and no tillage than soils with successional systems. Magnesium was higher in soils with no tillage and conventional tillage than soils with successional systems. Potassium was higher in soils with no tillage and conventional tillage than soils with successional systems. Base saturation percent of Calcium was highest in soils with conventional tillage, followed by the soils with no tillage and successional systems. Base saturation percent of Magnesium, Potassium, and Sodium were higher in soils with no tillage and conventional tillage than soils with successional systems. Base saturation percent of other bases and Hydrogen were higher in soils with successional than no tillage and conventional tillage. Boron and Copper were similar in soils with conventional tillage, no tillage and successional systems. Iron was higher in soils with successional and no tillage than soils with conventional tillage. Manganese was highest in soils with conventional tillage system, followed by the soils with successional and no tillage systems. Zinc was highest in soils with successional system, followed by the soils with conventional tillage and no tillage systems. Aluminum was higher in soils with conventional tillage than soils with successional and no tillage.
3. Soil nematode communities in soils with long-term conventional tillage (CT), no-tillage (NT) and successional (SC) systems: Bacterial feeding nematodes were higher in soils with successional and conventional tillage than those with no tillage. Fungal feeding nematodes were very similar in all three systems with no significantly difference. Omonivorous nematodes were significantly different in three systems, and it was highest in soils with conventional tillage, followed by the soils with no tillage and successional systems. Predator and stunt nematodes were higher in soils with no tillage than soils with successional and conventional tillage. Lesion nematodes were highest in soils with successional system than soils with no tillage and conventional tillage. Stubby root, spiral and dagger nematodes were higher in soils with conventional tillage and successional than soils with no tillage system.
4. Burkholderia communities in soils with long-term conventional tillage (CT), no-tillage (NT) and successional (SC) systems: Burkholderia populations were characterized in soils with long-term tillage (CT), no-tillage (NT) and successional (SC) systems using a most probable number (MPN) microtiter plate assay based on 21 sampling points in each system. MPN assays demonstrated that Burkholderia populations were significantly higher in SC soils compared to CT and NT soils. SC sample sites were dominated with successional native species whereas CT and NT were planted to soybeans at the time of sampling. Blast searches and phylogenetic analysis based on the sequences of the 16S rRNA demonstrated different soil management practices modified Burkholderia species composition. The soils with CT consisted of uncultured Burkholderia species, the dominant species in this system, B. cepacia, B. glathei, B. caribensis, and B. terrae. The soils with NT consisted of uncultured Burkholderia species, B. cepacia, B. ambifaria, B. cenocepacia, B. anthina, B. caribensis and B. terrae, with an even species distribution. The SC soils consisted of uncultured Burkholderia species, B. caribensis, B. terrae, and B. ambifaria, with B. caribensis, B. terrae, and uncultured Burkholderia species as the dominate species. In addition, denaturing gradient gel electrophoresis is in progress to characterize Burkholderia species diversity. This research demonstrated that the soil management practices affected the quantity and composition of Burkholderia spp.
5. Pythium communities in soils from long-term conventional tillage (CT), no-tillage (NT) and successional (SC) systems: Pythium populations were characterized in soils with long-term conventional tillage (CT), no-tillage (NT) and successional growth (SC) using dilution plating and colony counts. Analysis of 21 samples using a grid sampling design demonstrated Pythium populations were significantly higher (P = 0.05) in SC plots (18.5 cfu/g dry weight soil) than NT levels (11.4 cfu/gdw) with CT numbers the lowest (7.3 cfu/gdw). SC sample sites were dominated with successional native species whereas CT and NT were planted to soybeans at the time of sampling. Individual species were identified using BLAST search of the internal transcribed spacer 1 (ITS1) of the representative isolates (n=21/system). The soils with CT consisted of P. spinosum, P. irregulare and P. ultimum, and the soils with NT consisted of P. spinosum, P. irregulare and P. attrantheridium, respectively. P. spinosum was the dominant species in CT and NT systems. The SC soils consisted of P. spinosum, P. irregulare and P. attrantheridium, with P. irregulare and P. attrantheridium as the dominant species. In addition, denaturing gradient gel electrophoresis is in progress to characterize Pythium species diversity. This research demonstrated that the soil management practices affected the quantity and composition of Pythium spp.
6. Fusarium communities in soils from long-term conventional tillage (CT), no-tillage (NT) and successional (SC) systems: Fusarium spp. were also influenced differentially by the three management practices. The Fusrium population was significantly higher in the CT and NT systems with a mean number of colony forming units of 521 and 514 compared to 458 in the SC system (P=0.05). DGGE analysis and sequencing of a fragment f the Elongation factor gene enabled identification of many Fusarium species. Some Fusarium spp. such as F. oxysporum, F. solani, F. equiseti, only existed in CT and NT soils; F. moniliforme, and F. graminearum only existed in NT soils. However, other species such as one genotype of F. oxysporum, F. langsethiae and F. redolens were not affected by the different management practices.
7. Functional soil assays were conducted to determine the incidence of damping off symptoms on beans when planted in the soils from the different farming systems. All 63 samples were assayed independently and pathogens were isolated and characterized from root lesions. NT incited the highest level (P=0.05) of disease incidence (rating = 0.97) compared to CT (rating =0.91) and a much lower incidence of 0.67 in CT soils. The incidence and species composition of pathogens isolated were distinct from each farming system soil.
8. Canonical correspondence analysis and regression analysis was used to determine the relationship of soil physical, chemical and biological parameters with farming system. Surprisingly, these three broad attributes were highly distinct in each farming system (except the texture of sand/silt/clay content was similar for the framing system sites). These data showed that farming systems over a ten year time frame dramatically impacted soil physical, chemical and biological components of the soil. The challenge now is to link this data with a comprehensive microbial community 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). To test the impacts of soil microbial diversity on the stability of plant systems, we examined how microbial diversity affected the population dynamics and activities of Pythium ultimum in nine reconstructed soils along a microbial diversity gradient. The diversity gradient was created through reciprocally introducing microbial communities from three soils with significantly different microbial activities and diversity. This also allows us to isolate the effects of resource availability and diversity as both are interconnected in nature. We characterized soil microbial diversity with Biolog, fatty acid profile and microarray of functional genes (data under analysis). We predicted that the density and activity of the introduced pathogen would be lowest in soils with the highest diversity. Surprisingly, highest population densities occurred in highly diverse soils, which contradicts the stated prediction. Pathogen densities were nearly 4-5 times larger in the species-rich than the species-poor soils. However, the activity of the pathogen, measured by seedling mortality of a susceptible host tomato, was lowest in the most diverse soils. Pythium populations significantly correlated to soil labile C and microbial biomass C, but pathogen activity was negatively related to these two parameters.

Accomplishments/Milestones

Soils were re-sampled from the long-term farming systems experiment at the Center for Environmental Farming Systems Research Site (Goldsboro, NC) in the fall of 2007. The sampling occurred in three “farming systems” to look at the spatial dynamics of microbial communities from 1 to 10000 sq. m collected using a systematic sampling design.
Homogenized and pooled soil samples representative of selected distances 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. Data collected and analyzed to date include soil physical and chemical parameters, portions of the microbial community (e.g. Burkholderia sp.), Pythium and Fusarium population levels, damping off incidence in functional seedling assays and diversity and nematode community analysis. Multivariate analysis has been initiated to learn about the relationships between the parameters measured. A series of experiments were conducted to assess the impact of microbial abundance or microbial diversity on disease suppression using tomato seedlings as an indicator crop.

Impacts and Contributions/Outcomes

We anticipate the bulk of the impacts and outcomes will come when all the data has been assimilated and subjected to detailed statistical analysis. To date, it is clear that farming systems have had a large impact on soil parameters, many of which are considered “soil health” indicators. With the systematic sampling design used and the polyphasic analysis of the soil samples for physical, chemical and biological parameters we expect to learn about key links between components that growers can manage (e.g. carbon input and farming system), components that growers cannot manage (e.g. soil texture) and the impact on microbial communities, plant pathogens and ecosystem services.

Related presentations and outcomes from this work includes (2008-2009):

Louws, F.J., B. Liu and J.P. Mueller. 2009. Farming system impacts on plant pathogen and Burkholderia diversity and plant health. Bacterial Genetics and Ecology 10th annual Conference. Uppsala, Sweden.
Louws, F.J. and B. Liu. 2009. Integrated management of soilborne plant pathogens: From chemical based tactic substitution to microbial ecology based tactic development. Multitrophic Interactions in Soils, Uppsala, Sweden.
Liu, B and F.J. Louws. 2008. Burkholderia communities in soils with long-term tillage, no-tillage and successional systems. Phytopathology 98: S92.
Liu, B and F.J. Louws. 2008. Characterization of Pythium communities in soils from conventional tillage, no-tillage and successional systems. Phytopathology 98: S92.
Liu, B and F.J. Louws. 2009. Communities of Pythium and Fusarium in soils from conventional tillage, no-tillage and successional systems and their relationship with seed rot and damping-off of soybean. Phytopathology 99:Sxx.
Louws, F.J. Soil health workshop. Panel speaker. AUSVEG – 2009 Vegetable Industry Conference. Melbourne Australia. 5 May 2009.
Louws, F.J. Integrated management of vegetable diseases and soil health. Keynote speaker. AUSVEG – 2009 Vegetable Industry Conference. Melbourne Australia. 4 May 2009.
Louws, F.J. Tomato Grafting and High Tunnel Production: Pathology Problems and Horticultural Solutions. Department of Horticultural Science, NCSU. April 20, 2009.
Louws, F.J. Plant Disease Management: Unearthing Links Between Soil, Plants and Microbes. 6th International IPM Symposium. Portland Oregon. March 24, 2009.
Louws, F.J. “Fight Crop Disease: Soil Amendments and Biofumigation”. National Sustainable Agriculture and Research Education (SARE) 20th Anniversary “New American Farm Conference”. 26 Mar 2008. Kansas City MO. (SARE has funded over 3700 projects nationally and selected key people from that group to lead workshops. I was most honored to be invited. Attendance 800 people).

Collaborators: