Influence of microbial species and functional diversity in soils on pathogen dispersal and ecosystem processes in organic and conventional agroecosystems

Influence of microbial species and functional diversity in soils on pathogen dispersal and ecosystem processes in organic and conventional agroecosystems

Summary

Organic production systems have increased in recent years in many areas of the United States. Organic systems do not use synthetic pesticides and in the long term may be more sustainable than conventional systems. It is often assumed that soils from conventional agroecosystems will be more prone to pathogen invasion then soils from organic agroecosystems, since species diversity is greater in soils from organic agroecosystems. However, this hypothesis has never been critically examined in the field using spatial statistical modeling and dispersal gradient analysis with introduced plant pathogens. We know little about how to manage soil microbial communities to reduce pathogen invasion and optimize plant disease suppression and ecosystem level processes. Is it more important to manage functional or species diversity to enhance suppressiveness of soils to plant pathogens? Only a few comparative studies have been conducted to examine the relationship between plant disease suppression, soil physical and chemical factors and the biological communities present in soils from organic and conventional agroecosystems. None of these studies have been conducted on a spatial scale in the southeastern US.

This research began in the summer of 2001. A portion of the field research was conducted at the Horticultural Crops Research Station (HCRS) in Clinton, North Carolina in field plots that were established in the fall of 1996 on a previous SARE grant. The objectives of our research are to examine the influence of microbial species and functional diversity and composition on the invasion of soils from conventional and organic agroecosystems by the Basidiomycete plant pathogen Sclerotium rolfsii, causal agent of Southern blight. Specifically, we will contrast conventional soil fertility amendments including synthetic fertilizers, and organic soil fertility amendments, including either composted plant materials, animal manures, or incorporated green manures on the spread of this pathogen and the community dynamics of selected microflora in field plots. In on-farm tests, we will evaluate the resistance of conventional versus organic agrecosystems to species invasion and colonization by this plant pathogen. We will sample soils on both organic and conventional farms that have received either organic or conventional soil fertility amendments and compare disease suppressiveness of these soils and determine the functional components of soil microbial communities associated with disease suppression. In a third field experiment, we will determine whether withdrawal of specific pesticide components during transition from conventional to organic production systems impacts soil microbial communities and subsequent disease caused by S. rolfsii. We plan to enhance our current education efforts at N. C. State University by teaching several new classes including a course in Agriculture, Ethics, and the Environment, and one in Soil Ecology. The Statistics Department offers courses in Applied Multivariate and Spatial Statistics. We plan to continue our educational outreach efforts at grower meetings and on-farm meetings conducted during this project.

Objectives/Performance Targets

Objective 1. Are soils from organic agroecosystems more resistant than soils from conventional agroecosystems to invasion by the soilborne plant pathogen S. rolfsii? How do the spatial patterns of disease symptom expression relate to pathogen propagules of S. rolfsii in soils in field plots from conventional and organic agroecosystems?

Objective 2: Are grower field soils from organic farms amended with organic fertility amendments more suppressive to disease caused by S. rolfsii than soils from conventional farms amended with synthetic fertility amendments? Is species diversity, functional diversity or the composition of the soil microflora most closely related to disease suppressiveness?

Objective 3: Does the strategy of transition from conventional to organic production systems impact species diversity, functional diversity and composition of microflora and microfauna in soils and the subsequent dispersal of the soilborne pathogen S. rolfsii?

Objective 4: Continue education and outreach plans by teaching graduate level courses relevant to sustainable agriculture research, conducting on-farm research and education, and presenting data at conferences with a focus on ecology in agriculture.

Accomplishments/Milestones

Objective 1. Are soils from organic agroecosystems more resistant than soils from conventional agroecosystems to invasion by the soilborne plant pathogen S. rolfsii?

This portion of the field research is underway at the Horticultural Crops Research Station (HCRS) in Clinton, North Carolina in field plots that were established in the fall of 1996 on a previous SARE grant. The experimental design consists of a split plot. The frequency of disturbance (tillage) during the growing season differs between the main plot treatments (2 levels) and includes either biweekly tillage on bare-soil plots or surface-mulch with wheat straw after a single initial tillage on mulched plots. The mainplots are arranged in 4 blocks. Subplots (4 levels) include: a conventional synthetic fertilizer, or organic fertility amendments including either a composted cotton-gin trash, composted poultry manure, or an incorporated rye-vetch green manure (cover crop planted in previous fall). Rates of each soil amendment were standardized to obtain 112 kg plant available nitrogen per hectare. Each experimental unit consists of six rows that are 7.6-m long and 1.6-m wide. Treatments are replicated four times. Six-week old pepper seedlings were transplanted 14 days after soil amendment in single rows at 30 cm within-row spacing, and overhead irrigation was utilized as needed through the season (2.5-3.0 cm per week without rain). Main plots received tillage biweekly until plants were too large for a tractor to clear. Wheat straw was applied to the other main plot treatments 2 weeks after transplanting.

Disease incidence, spatial dynamics, and yield

Final disease incidence in 2001 caused by southern blight in bell pepper ranged from 6.6 to 21.1 % (Table 1). There was high variability both within and among main plots; the subplot variance was 1.50 and the whole plot variance was 1.15. The standard error for each treatment mean was 8.14. Thus, there were no statistically significant effects of tillage or soil fertility amendments on disease. Overall disease incidence was low in 2001. However, some general trends were observed. Highest disease incidence occurred in tilled plots amended with synthetic fertilizers and lowest disease incidence occurred in surface mulched plots that were amended with either a rye-vetch green manure or a composted poultry manure. In general, disease increased to a greater extent over time in tilled than mulched plots (Fig. 1). In 2001, disease was high in block one of the experiment in plots that received composted cotton gin trash (Fig. 2). Many cotton growers rotate with peanuts and peanuts are highly susceptible to Southern blight. Pathogen sclerotia may have been present in the compost we used this year. On the other hand, soil moisture may have been high in this low area of the field in block one and this may have affected disease (Fig. 2). We are currently evaluating both of these possibilities. Overall, disease incidence in bell pepper was lower than in previous years when the field was planted to processing tomato. Pepper may be less susceptible to southern blight since stems are more upright than those of processing tomatoes and thus contact with sclerotia may be less likely.

How do the spatial patterns of disease symptom expression relate to pathogen propagules of S. rolfsii and soil moisture in soils in field plots from conventional and organic agroecosystems?

The center four rows of the six row plots were divided into 1m quadrats containing 3 plants and coordinates were determined and marked with the use of a global positioning system. Each treatment and rep combination (32) contained 28 quadrats and a total of 896 quadrats were monitored for disease across the field (Fig. 2). The spatial and temporal dynamics of disease spread were evaluated over time by monitoring the incidence and severity of infected plants in each quadrat. Disease severity was recorded on individual plants on a quadrat basis, two times per week for a period of five weeks. We have just hired a graduate student in statistics to conduct the spatial statistical analysis of disease spread within the field (Fig. 2). A preliminary map of final disease incidence in each block has been developed but analysis of the spread of disease over time has not been conducted yet to determine whether there were differences in the spatial dynamics of disease spread among plots amended with either conventional or organic fertility amendments. Soil from each quadrat was also assayed for soil water content and population densities of sclerotia of S. rolfsii. Population densities of sclerotia were below detectable levels in the field.

Soil fertility amendments had a significant effect on yield. Highest yields occurred in plots amended with cotton gin trash or synthetic fertilizer (22.4 and 18.1 kg/per plot, respectively) and lowest yields occurred in plots amended with either the rye-vetch green manure or composted poultry manure (6.5 and 7.4 kg/ plot, respectively). There was no significant relationship between yield and disease incidence since highest yields occurred in plots that had the highest level of disease (Table 1). Overall disease incidence was low in all the plots and fruit set and yield were not compromised.

Soil fertility amendments had a significant effect on all the chemical factors that were measured in the plots with the exception of % humic matter. Cation exchange capacity was highest in soils amended with cotton gin trash. The base saturation and pH of soils amended with the organic fertility amendments were also higher than soils amended with synthetic fertilizer. In general, P, K, Ca, Mg, and Mn levels were higher in soils with compost amendments than synthetic fertility amendments. Soils amended with composted poultry manure had higher levels of copper and zinc than soils in the other plots.

Soil microbial communities, microbial biomass, respiration, physical and chemical factors

Population densities of thermophilic organisms (grow at temperatures > 40 C) were significantly higher in plots amended with composted cotton gin trash than in the other plots (Table 2). Population densities of thermophiles were 1.1 x 103 colony forming units (cfu) per gram dry soil in plots with composted cotton gin trash and 4.4 x 102, 3.4 x 102 and 8.3 x 102 cfu/g dry soil in soils amended with synthetic fertilizers, composted poultry manure or a rye vetch green manure, respectively. Population densities of total fungi (6.1 x 104 versus 4.1 x 104 cfu/g dry soil) and beneficial fluorescent Pseudomonas species (9.8 x 105 versus 5.5 x 105 cfu/g dry soil) were higher in mulched than tilled soils (Table 2). Population densities of the beneficial biocontrol fungi in the genus Trichoderma were not different among treatments. There were also no differences in population densities of total culturable bacteria or enteric bacteria among treatments.

Microbial biomass carbon (MBC) was significantly higher in mulched than tilled soils in early stages of plant growth, but this trend reversed later in the growing season (Table 3). In August, the highest MBC was found in tilled plots amended with cotton gin trash (CGT), while the lowest MBC was in tilled plots amended with rye-vetch (RV) or synthetic fertilizer (SF) (Fig. 3A). Microbial biomass nitrogen (MBN) was not different between mulched ant tilled plots early in the season (Table 3), but was significantly higher in plots amended with cotton gin trash than the other soil amendments in August (Table 3, and Fig.3B).

The microbial biomass C to N ratio was higher under mulch than tillage in May, but not different afterwards (Table 3). Cotton gin trash addition significantly increased the microbial biomass C to N ratio in May (Table 4), but microbial biomass C:N ratio in all soils decreased to about 5 in August. These findings suggested that growing plant roots may have offset the effects of mulching and amendments on this ratio.

Soils amended with cotton gin trash or poultry manure also had higher soil respiration rates than plots amended with synthetic fertilizers or rye vetch green manure (Fig. 3C). Soils from tilled plots amended with cotton gin trash had the highest respiration rates while tillage had less of an impact in plots amended with synthetic fertilizers, poultry manure or rye vetch (Fig. 3C).

Mulched soils had reduced net N mineralization in May and August, but increased net nitrogen mineralization in June, as compared to tilled soils (Table 3). Soils amended with cotton gin trash had increased net N mineralization and available N, while soils amended with poultry manure, rye vetch or synthetic fertilizer had decreased N mineralization and reduced available N to plants (Fig 3D).

Objective 2: Are grower field soils from organic farms amended with organic fertility amendments more suppressive to disease caused by S. rolfsii than soils from conventional farms amended with synthetic fertility amendments?

We are interested in asking the question “Will soils from organic grower fields with a history of organic fertility amendment use be more suppressive to pathogen dispersal, sclerotia germination and disease then soils from conventional fields amended with synthetic fertilizers?” These studies will provide quantitative data on spatial and temporal components of pathogen invasion and disease spread in soils from contrasting conventional and organic agroecosystems. In late summer of 2001, 20 kilograms of soil was removed in a uniform pattern from 3 sites at each of 5 organic (Dawson, Hartmann, Harman, Hitt, Letendre) and 5 conventional (Hope, Holland, Hall, Cottle, HCRS-Clinton) fields. The organic farms used cover crops and organic soil amendments (composted animal manures, feather meal or yard waste) and the conventional farms used synthetic fertilizers for plant nutrition. Two undisturbed cores were removed from three locations at each farm for bulk density and soil water release measurements. Soil cores were removed from three areas at each farm to a depth of 8 inch and bulked. Soil is currently being tested in greenhouse assays for disease suppressiveness to Southern blight. Sclerotia viability will be determined by methanol assay. Spatial development of disease will be assayed in subsequent greenhouse experiments. The greenhouse disease assays are in progress at this time. Our observations in 2001 indicate that the conventional growers had a history of problems with Phytophthora blight in vegetables on their farms, while the organic growers had low incidence of southern blight in vegetables on their farms. Thus, the specific disease problems vary between production systems.

Is species diversity, functional diversity or the composition of the soil microflora most closely related to disease suppressiveness?

Culture dependent assays were used to measure the relative abundance of various microbial populations using 10-fold serial dilutions of soil and selective media. Numbers of culturable bacteria, total fungi, and thermophilic microorganisms were quantified. In addition, numbers of species of Trichoderma and fluorescent Pseudomonas species were assessed since they have been associated with suppressive soils and are known biological control agents or have growth-promoting effects on plants. In addition, enteric bacteria were quantified. Paired t –tests were conducted to compare soil microbial populations from organic and conventional fields. Surprisingly, population densities of the organisms in soil that we quantified were not statistical different between organic and conventional farms (Table 5). The abundance of total culturable fungi and bacteria, thermophiles, and fluorescent Pseudomonas species were higher in soils from organic than conventional farms. However, only one late summer sample has been taken on the farms at this point in time.

Total and active fungal and bacterial biomass was also measured in the soils from organic and conventional farms. There were no differences in total fungal biomass in soils between organic and conventional farms (Fig. 4A). However, there was variation among both organic and conventional farms in the level of fungal biomass present in soils. (Fig 4A). Conventional farms had significantly higher (P = 0.03) bacterial biomass than organic farms (Fig. 4B, Table 6). The ratio of active to total fungal biomass was not different between soils from organic and conventional farms (Fig. 4C). However, the ratio of active to total bacterial biomass was significantly greater (P = .002) in soils from organic than conventional farms (Fig. 4D)

Variations in population densities and microbial biomass of the soil organisms may be closely related to the production practices used in individual fields. One organic grower was a greenhouse tomato producer and solarized his soil, whereas one conventional grower used methyl bromide to fumigate the previous fall. Both of these growers had reduced fungal populations and fungal biomass compared to the other soils. We have developed a field survey and sent it to the 10 grower collaborators to learn more about the individual production practices and cropping histories on the farms. This data will be used in multivariate statistical analysis with other soil indicators.

Microbial biomass C (MBC) ranged from 172 to 1720 mg kg-1 in soils from organic farms (mean = 603 mg kg-1) and from 61 to 251 mg kg-1 in soils from conventional farms (mean = 133 mg kg-1) (Fig. 5A). Higher labile C was recorded in some soils from organic farms (Fig. 5B), leading to higher microbial activity (CO2 evolution) in soils from those farms (Fig. 5C) than conventional farms.

Organic growers had significantly greater levels of manganese, zinc, and sodium in their soils than conventional growers (data not shown). Percent humic matter, CEC, % base saturation, % base acidity, pH, potassium, phosphorus, calcium, magnesium, copper and sulfur also were higher in organic than conventional soils, but these differences were not statistically significant.

Three subsamples of each soil from each site at the five conventional and five organic grower locations were immediately stored in a freezer (-20 C) and will be used to assess the microbial community structure by analyzing the phospholipid fatty acid (PLFA) biomarkers. Genetic diversity of the fungal and bacterial communities in soil will be assessed using DNA directly extracted from soil and amplified with primers in a polymerase chain reaction. The above assays have not been conducted yet. We are in the process of hiring a postdoctoral associate that will conduct this aspect of the research. Community level physiological profiling (CLPP) was conducted within 3 weeks after sampling by examining sole-C substrate use patterns of the microbial communities on Biolog microplates. We are in the process of analyzing the substrate utilization pattern data and will report this at a later time.

Objective 3. Does the strategy of transition from conventional to organic production systems impact species diversity, functional diversity and composition of microflora and microfauna in soils and the subsequent dispersal of the soilborne pathogen S. rolfsii?

Does the strategy of transition from conventional to organic production systems differentially impact the communities of microflora and microfauna that sucessionally recolonize soils after pesticide withdrawal? Are there optimal pesticide withdrawl strategies during the transition to organic production that can enhance soil biological diversity and reduce disease? This experiment is underway at the Center for Environmental Farming Systems (CEFS) field site in Goldsboro, NC and is part of a larger farming systems project underway at CEFS (Paul Mueller, Project coordinator, USDA Southern SARE grant LS01-120).

The experimental design consists of a randomized complete block where specific pesticidal inputs were selectively withdrawn over time from field plots compared to an organic (no pesticides) or conventional (pesticides applied) system. This design includes six, 0.1-hectare treatments per replication. One treatment (conventional) consists of conventional inputs including pesticides and synthetic fertilizers. Treatment two (immediate organic) consists of an organic treatment with no synthetic pesticides or fertilizers. Treatments 3 to 5 (selective) include selective withdrawals of either fertilizer, herbicide or pesticide over a three-year period. Treatment 6 (gradual) includes a gradual withdrawal of pesticides and synthetic fertilizers over a 3-year period. After three years, all four transitional strategies will have substituted organic inputs for all conventional inputs in treatments 3 to 6.

Culture dependent assays were used to measure the relative abundance of various microbial populations using 10-fold serial dilutions of soil and selective media. Numbers of culturable bacteria, total fungi, and thermophilic microorganisms were quantified. In addition, numbers of species of Trichoderma and fluorescent pseudomonads were assessed since they have been associated with suppressive soils and are known biological control agents or have growth-promoting effects on plants. Soil was sampled in late summer 2000 and 2001 from the same spatially referenced areas in the plots. Five sample points were sampled within each experimental unit in 2000 and three of five spatially referenced points were sampled in 2001. No significant differences were found between treatments for any of the microbial populations measured in 2000. In 2001, population densities of total fungi were significantly higher in treatment 6, where pesticide levels were gradually withdrawn over the two year period. Population densities were 7.7 x 104 cfu/g of dry soil in the gradual reduction plots, while population densities were 3.8 x 104 cfu/g of dry soil in soils from conventional plots, and 2.9 x 104 cfu/g of dry soil in soils from organic plots where pesticides were not used at all. This experiment is in its second year of transition, and the magnitude of differences observed was very small. There were also no statistical differences in germination or parasitism of sclerotia of S. rolfsii that were placed in soil retrieved from these plots.

Impacts and Contributions/Outcomes

The Project coordinator has been involved in research and outreach at the Horticultural Crops Research Station (HCRS) in Clinton, NC since 1987 and has taught soil quality workshops and participated in numerous field days for county agents and growers at many locations within North Carolina and at commodity group meetings across the southeast. This year the PI and her research technician attended the Carolina Farm Stewardship meeting in Rock Hill South Carolina and discussed data with county agents and organic growers in the southeastern US. In addition, the PI visited farms of all the grower collaborators on the project and learned about their operations. A meeting is planned for early spring to share data with both the organic and conventional growers. We are working with county agent Debbie Roos in Chatham County, NC and Alan Thornton in Sampson County, NC to organize farm visits and grower meetings. A farm survey has also been developed and sent to the growers to gather more information on their farming practices and history of plant disease.

The PI and co-PI (Hu) were involved in teaching a soil biology workshop for undergraduate summer interns in Sustainable Agriculture that began at the Center for Environmental Farming Systems during the summer of 2000. The co-PI (Hu) organized a new class in Soil Ecology that was taught for the first time in the fall of 2001. The project coordinator will teach a class called “Agriculture, Ethics and the Environment” (http:www.cals.ncsu.edu/course/pp590a) in the fall of 2002 in the Department of Plant Pathology at N. C. State University. Students enrolled in the class have included graduate students from many departments in CALS, county agents, North Carolina Dept. of Agriculture pesticide administrators, N.C. Department of Environment and Natural Resources statisticians, and growers. Written reviews from students who have taken the class have been excellent. The class is taught every other year and will be taught again in the fall of 2002. Students use role playing and case studies to discuss issues such as populations and food, ozone depletion, animal waste management, water quality, intellectual property rights and the third world, genetically modified crop plants, organic and sustainable agriculture, pesticide resistance development, and research ethics. The other co-PI (Gumpertz) will teach classes in multivariate and spatial statistical techniques.

The PI attended the Organic Farming Research Foundation sponsored First Scientific Congress on Organic Agriculture Research in January 2001 in Monterey, CA and a portion of the 2nd SCOAR Conference in November 2001. She presented a poster on some of the data collected from a previously funded SARE research study (LS95-70) and the current SARE funded project (LS01-128) on organic farms in the southeast. This meeting brought research scientists, growers and policy makers to the table to discuss research issues that are important for organic production systems. The PI and co-PI (Hu) are also members of the Ecological Society of America and attended and presented data from previous SARE funded research at the ESA meeting in Madison, WI in August of 2001.
Two publications are now in press and one is in review from the previous SARE funded project conducted at the Horticultural Crops Research Station, the Center for Environmental Farming Systems, and grower fields including:

Bulluck, L. R., and Ristaino, J. B. 2002. Synthetic and organic amendments affect Southern blight, soil microbial communities and yield of processing tomatoes. Phytopathology 92:181-189.

Bulluck, L. R., Evanylou, G. K., and Ristaino, J. B. 2002. Influence of Alternative and Synthetic Soil Fertility Amendments on Soil Microbial Communities and Physical and Chemical Properties on Organic and Conventional Farms. Appl. Soil. Ecol.19:147-160.

Bulluck, L. R., Barker, K. R., and Ristaino, J. B. 2001. Nematode trophic group and community dynamics on tomato as influenced by organic and synthetic soil fertility amendments. Appl. Soil Ecology: submitted.

Collaborators: