Final Report for LS01-128
The objectives of our research were 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 contrasted 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 S. rolfsii and the community dynamics of selected microflora in field plots. We evaluated the resistance of conventional, sustainable and organic agrecosystem soils to species invasion and colonization by S. rolfsii. Soils amended with organic amendments such as cotton gin trash were more suppressive to southern blight than soils from plots amended with synthetic fertilizers. These soils were colonized less rapidly over time by the pathogen and disease incidence was reduced and yield was higher at harvest. Soils with organic amendments had higher populations of thermophilic organisms. Grower field soils from conventional, organic and sustainable farms were sampled and assayed for disease suppressiveness in greenhouse assays. Soils from organic farms were more suppressive to disease than soils from sustainable or conventional farms in each season. These soils contained bacterial communities that were more diverse than soils from conventional farms. Soils from organic farms had higher levels of extractable C and N, higher microbial biomass carbon and nitrogen, and net mineralizable N. In addition, soil microbial respiration was higher in soils from organic than sustainable or conventional farms, indicating that microbial activity was greater in these soils. We are currently examining the functional and species components of soil microbial communities associated with disease suppressiveness.
In this research we addressed the following objectives:
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. 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.
Agricultural ecosystems are highly vulnerable to species invasion by plant pathogens. Billions of dollars worth of agricultural chemicals are applied annually to crops to prevent species invasion by pathogens, insects, and weeds. Most plant systems in agriculture are highly uniform and monoculture is the norm. Fumigation of soils with fumigants such as methyl bromide reduces species diversity in soil and can make systems more vulnerable to pathogen invasion. Pesticide resistant strains of insects, weed, plant and animal pathogens pose risks to human health and the environment.
Organic production has increased tremendously in the United States in recent years and methods of environmentally sound disease control in these production systems are needed. The “Green Revolution” was highly dependent on synthetic fertilizers. In the 1990’s the demand by consumers for organically grown produce increased 24% yearly. Synthetic pesticide residues and genetically modified (GM) food crops have caused concerns for many consumers particularly in Europe. Since neither GM crop plants nor synthetic fertilizers are permitted in organic production systems, the produce is more desirable to consumers. This has brought about another type of “Green” revolution in the United States agricultural community.
Biodiversity has become a subject of much interest in recent years as permanent loss of habitats and individual species occurs. For many soil microorganisms, species may be lost before their identity and function are even realized. Studies of the biocomplexity of the microflora and microfauna present in soil can be studied at many spatial scales, but the scale of interest for production of healthy crop plants is around individual plant roots and between plants in the field. Studies of soil biocomplexity from contrasting organic and conventional agroecosystems at these scales will improve our understanding of ecological processes in soils important for sustainable crop growth and disease control.
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 of microflora and microfauna present in soils from organic and conventional agroecosystems.
Objective 1. Are soils from organic agroecosystems more resistant than soils from conventional agroecosystems to invasion by the soilborne plant pathogen S. rolfsii?
Experimental design: This 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 soil texture was a loamy sand (84% sand, 8% silt and 7% clay). The experimental design was a split plot. The frequency of disturbance (tillage) during the growing season differed between the main plot treatments (2 levels) and included either weekly tillage on bare-soil plots or surface-mulch with wheat straw after a single initial tillage on mulched plots. The main plots were arranged in 4 blocks. Subplots (4 levels) included: a conventional synthetic fertilizer (10-10-10 applied at 400-600 lbs/A), or organic soil fertility amendments including either composted cotton-gin trash, composted poultry manure, or an incorporated rye-vetch cover crop. Rates of each soil amendment were standardized to obtain 112 kg plant available nitrogen per hectare. Cotton-gin trash and poultry manure contained 8.1, 2.3, 3.9, and 0.25 lbs/ton and 19.1, 18.2, 21.9 and 2.2 lbs/ton of plant available nitrogen, phosphate, calcium and sodium, respectively. The rye/vetch cover crop was incorporated into subplots in early May of 2002 and late April 2003. The same plots and treatments have been in place for the last 7 seasons. Each experimental unit consisted of six rows that were 7.6-m long and 1.6-m wide. Treatments were replicated four times. Six-week old tomato seedlings (cv Rio Colorado) 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 that received tillage were tilled biweekly until plants were too large for a tractor to clear. Plots that received wheat straw were tilled twice before straw application approximately 2 weeks after transplanting. The field was naturally infested with S. rolfsii.
Disease Incidence and Yield: The interior four rows of each experimental unit were monitored weekly for symptoms of southern blight. In 2002, disease was monitored from June 19 until August 16, and in 2003, from June 27 until August 18. Disease severity was rated as: 0-healthy; 1-chlorosis, with or w/o a lesion; 2- wilt, lesion present; 3-severe wilt, dropped leaves, lesion present; 4-dead; 5-missing. The four interior rows were harvested from each experimental unit and the yield of green, yellow and red tomatoes was determined.
Statistical Analysis: The Statistical Analysis Systems software (PC-SAS 8.0; SAS Institute, Cary, NC) was used for data analysis. A generalized linear model procedure, a mixed model and analysis of variance (ANOVA) were performed for final disease incidence and tomato yield data. The area under the disease progress curve (AUDPC) was calculated and spatial maps of disease incidence in each year were made.
Soil microbial communities, microbial biomass, respiration, carbon and nitrogen, physical and chemical factors
Soil water content was measured gravimetrically and a soil water characteristic curve was determined for soil from each treatment in sieved (10-mm) soil at soil water matric potential values between 0 and -30.0 kPa with either Büchner tension funnels or a pressure plate apparatus (Hillel, 1982). Soil samples taken from each plot were sent to a soils testing laboratory to determine pH, texture, % base saturation, CEC, and soil electrical conductivity. In addition, the soil was tested for levels of inorganic N, total N, and organic C, soil Ca, Mg, Zn, Cu, Mn, K, Al, P, and Na with the Mehlich-3 extractant and either atomic absorption spectrophotometry or flame emission (K, Na). Soil respiration, microbial biomass carbon, nitrogen, extractable N and carbon and net mineralized nitrogen were determined in each plot.
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 Gliocladium 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.
Community level physiological profiling (CLPP) was used to examine sole-C substrate use patterns of the microbial community on Biolog microplates. Total and active bacterial and fungal biomass was estimated by direct counts. Total microbial biomass was quantified using direct fumigation-extraction and KCl-extractable N was quantified. Microbial activity was assessed by quantifying respiratory activity and by fluorescein diacetate (FDA) hydrolytic activities. Available C per unit of microbial biomass was calculated and used as an index for C competition intensity. Potential C availability and water soluble C was assessed by quantifying CO2 evolution in incubation experiments at constant moisture and temperature conditions.
Final disease incidence in 2002 caused by S. rolfsii in tomato ranged from 32-50%. Soil fertility amendments significantly affected the incidence of disease, the area under the disease progress curve, and yield (Fig. 1A). Disease was significantly reduced by organic soil fertility amendments (Table 1). Disease incidence and the area under the disease progress curve were lowest in plots amended with cotton gin trash and highest in plots amended with synthetic fertility amendments. Yield was significantly affected by tillage and amendment. Yields were highest in tilled plots amended with cotton gin trash or poultry waste and lowest in straw-mulched plots amended with the rye vetch green manure (Table 1).
Disease onset occurred earlier and was more severe in 2003 (Fig. 1B). Final disease incidence reached 100% in most of the plots and the amendment effect was not significant. Disease incidence in both years was higher in tilled than straw-mulched plots (Fig. 2A,B), however, differences were not significant. In 2003, tillage had a significant effect on yield and mean yield was 4.9 lb/plot in tilled plots and 18 lb/plot in straw-mulched plots (Table 1). S. rolfsii spread more rapidly in soils from the conventional plots amended with synthetic fertilizers (Fig. 3A). Pathogen spread was greatly reduced in plots amended with cotton gin trash (Fig. 3B).
Soil microbial communities, microbial biomass, respiration, carbon and nitrogen, physical and chemical factors
Population densities of thermophilic organisms (organisms that grow at temperatures > 40 C) were significantly higher in soils amended with composted cotton gin trash than in the other soils. Population densities of thermophiles were 31,000 colony-forming units per gram of dry soil (cfu/g dry soil) in plots amended with composted cotton gin trash and 16,000, 10,000 and 12,000 cfu/g dry soil in soils amended with synthetic fertilizers, composted poultry manure or a rye vetch green manure, respectively. Population densities of oomycete fungi were also higher in soils amended with cotton gin trash (220 cfu/g dry soil) than soils amended with synthetic fertilizers (11 cfu/g), composted poultry manure (66 cfu/g dry soil) or a rye vetch green manure (140 cfu/g), respectively. There were no differences in population densities of total fungi, Trichoderma or Gliocladium species, culturable bacteria or enteric bacteria among treatments.
Soil bulk density was lowest in plots amended with cotton gin trash and soil moisture was highest in these plots. Tillage and amendments significantly influenced soil extractable carbon (C). Compared to tillage, straw-mulched plots had higher soil extractable C in both years (Table 2). Highest extractable C (64-88 mg C kg –1 and 47-61 mg C kg-1) was found in plots amended with cotton gin trash, followed by poultry manure (42-68 mg C kg-1 and 48-50 mg C kg-1), rye vetch (20-38 mg C kg-1 and 32-37 mg C kg-1) and synthetic fertilizer (17-29 mg C kg-1 and 32-40 mg C kg-1 ) in 2002 and 2003, respectively. The tillage and amendment effects for extractable C were significant in both years. Soil extractable N was higher in plots amended with poultry manure or cotton gin trash, than rye/vetch or synthetic fertilizers in both years and the amendment effect was significant (Table 2).
Microbial biomass C (MBC) was significantly higher in plots with straw mulch than in tilled plots in both years. Amendments also consistently influenced MBC. The largest microbial biomass C was found in the plots amended with cotton gin trash (261 to 517 mg kg-1) followed by the poultry manure (120 to 270 mg kg-1), rye vetch cover crop (114 to 278 mg kg-1) and synthetic fertilizer (98 to 208 mg kg-1). The MBC was significantly correlated with extractable C, microbial biomass nitrogen (MBN), and microbial respiration, and in most cases with extractable N and net mineralizable N.
There was significantly higher microbial biomass N in straw-mulched than tilled soils in 2002 and 2003. (Table 2). Amendments also significantly influenced MBN. The MBN was highest in soils amended with cotton gin trash followed by poultry manure, rye vetch and synthetic fertilizers. The MBN was significantly correlated with extractable C and MBC, and in most cases with extractable N, microbial respiration and net mineralizable N. Organic amendments and straw mulch did not influence microbial biomass C to N ratios in most cases (Table 2).
Soil microbial respiration was higher in straw-mulched than tilled plots (Table 2). Soil microbial respiration was also higher in plots amended with cotton gin trash and poultry manure than synthetic fertilizer or rye vetch. Microbial respiration was significantly correlated with MBC, MBN, NMN, and extractable- C for most sample dates. Surface application of straw mulch led to higher net nitrogen mineralization (potential N availability) than did tillage. Organic amendments had significant effects on N mineralization. Highest net mineralized N was found in plots amended with cotton gin trash, while plots amended with rye vetch, poultry manure and synthetic fertilizers released much less N. No significant interactions were observed between mulch and amendments for nitrogen mineralization. Net mineralizable N was significantly correlated with extractable C, and in most cases to MBC, MBN, extractable N and microbial respiration.
Soil fertility amendments had a significant effect on all the chemical factors that were measured in the plots with the exception of zinc, copper and % 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.
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?
In spring of 2002 and 2003, 20 kilograms of soil were removed using a 1-inch soil augur in a uniform pattern from three contiguous sites at three organic farms (Hartmann, Harmon, and Dawson), three sustainable farms (Hitt, Letendre, HCRS-Clinton), and four conventional farms (Hope, Holland, Hall, and Cottle.) Soil cores were removed from three areas at each farm to a depth of eight inches and bulked. Two undisturbed soil cores were removed from three locations at each farm for bulk density and water release measurements. The three organic farms used cover crops and organic soil fertility amendments for plant nutrition: composted animal manures, feather meal, or yard waste. The four conventional farms used synthetic fertilizer. The sustainable farms used a combination of synthetic fertilizers and composted plant materials.
Greenhouse assays. The experimental design was a randomized complete block with three replicates per farm. Inoculum of S. rolfsii was grown on sterile oat grains for 2 wks. Five oat grains were added to the center of each 4-inch pot filled with soil from the farms. The oats were pushed below the soil surface and pots were watered from above for two weeks before planting peppers. Five 4-wk-old peppers seedlings (variety Camelot) were transplanted into pots. Plants were observed weekly and disease incidence was recorded. The pathogen was isolated to confirm infection.
In 2002, disease incidence recorded from greenhouse assays was higher in soils from conventional farms in both trials (16.7%, and 21.7%) than in soils from either sustainable (4.5%- and 8.86%) or organic farms (15.56% and 11.1%) Similar results were recorded in 2003, however disease incidence was much higher. Disease incidence was highest in soils from conventional farms in both trials (51.7% and 60.0%) than in soils from either sustainable (39.96% and 24.43%) or organic farms (51.1% and 44.4%). Mycelial growth of S. rolfsii in soil was not different in soils from the different production systems in 2002 or 2003.
Is species diversity, functional diversity or the composition of the soil microflora most closely related to disease supressiveness?
Culture dependant assays were used to measure the relative abundance of various microbial populations using ten-fold serial dilutions of soil on selective media. Numbers of culturable bacteria, enteric bacteria, total fungi, Phytophthora and Pythium species, and thermophilic microorganisms were quantified. In addition, numbers of Trichoderma and florescent 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. The abundance of total culturable bacteria, and enteric bacteria were not significant in any year. Total culturable fungi were higher in soils from organic farms. Pythium and Phytophthora species were also highest in soils from organic farms all three years, but differences were not significant in 2003. Thermophilic organisms were significantly higher in soils from organic and sustainable, than conventional farms in 2002 and 2003 (Table 3).
Total and active fungal and bacterial biomass was also measured in soils from organic, sustainable, and conventional farms in fall 2001 and spring 2002 and 2003. Total fungal biomass was lower in soils from conventional than organic and sustainable farms in 2 of 3 years (Table 4). Total bacterial biomass was higher in soils from conventional than sustainable and organic farms for all three years of the study. Interestingly, in both 2001 and 2003 (test not performed in 2002) bacterial feeding nematodes were highest in soil from conventional farms. Soils from sustainable farms had the highest active bacterial and fungal biomass and the highest ratio of active to total fungal and bacterial biomass in 2001 and 2002. Active to total bacterial biomass was higher in soil from organic and sustainable farms than in soil from conventional farms in each year.
Soil chemical and physical factors also showed differences according to grower type. Soils from organic farms had significantly greater levels of phosphorus, calcium, magnesium, sodium, manganese, zinc, and copper than soils from conventional farms (data not shown.) Percent humic matter, cation exchange capacity, % base saturation, and pH were also higher in soils from organic and sustainable farms than conventional farms. Soils from conventional farms had the highest bulk density for all three years.
Microbial biomass carbon (MBC) was higher in soils from organic, then sustainable and conventional farms. Extractable carbon, microbial biomass carbon (MBC), net mineralized nitrogen (NMN) and soil microbial respiration (SMR) were all higher in soils from organic and sustainable farms than in soils from conventional farms for all three years of the study. Extractable nitrogen was also higher in soils from organic and sustainable farms in 2001 and 2002, but highest in soil from sustainable farms in 2003 (Table 5).
Three subsamples of each soil from each site at the conventional, sustainable, and organic grower locations were immediately stored in a freezer (-20C). DNA was directly extracted from soil using a MO BIO kit. The soil bacterial and fungal community structure was analyzed using gradient gel electrophoresis (DGGE) and DNA sequencing methods. Bacterial communities in soils from organic, sustainable and conventional farms were analyzed using molecular methods. The soil management practices had a great impact on the diversity of these communities. The majority of the bacterial communities in the soil were composed mainly of uncultured species (99%). Culturable bacteria are thought to represent only a small portion of the total bacteria in the soil. In samples from the spring of 2002 and 2003, bacterial communities were similar in soils from farms with organic and sustainable practices, while bacterial communities in soils from conventional farms were distinct. There were no distinct clades in samples collected in the fall of 2001. A database search based on DNA sequences revealed that the majority of the dominant populations of bacteria detected in soils from organic farms were mainly divided into three groups, which included Firmicutes (Bacillus), Actinobacteria and Proteobacteria (such as Pseudomonas). However, the bacteria communities in soils from conventional farms were mainly Proteobacteria. Uncultured bacteria were also detected in soils from organic and conventional farms. Denitrifying Bacillus species were found in soil from some organic and conventional farms.
Bacteria communities were more diverse in soils from organic farms than in soils from conventional farms. Cluster analysis divided the bacteria communities into two distinct groups. Organic and sustainable farms formed one cluster and conventional farms formed the second group with the exception of the Cottle and Letendre farms (Fig. 6). Letendre utilized annual soil solarization to control disease, which may have led to less species diversity in soil despite other sustainable practices. Cottle fumigated soil with methyl bromide and inoculated soil with a commercial mixture of beneficial microbes. Certain microbes recolonize soils quickly after fumigation and inoculation with known beneficials, may have significantly changed the microbial community.
Educational & Outreach Activities
Outreach. 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.
The project coordinator currently teaches a class called “Agriculture, Ethics and the Environment” (http:www.cals.ncsu.edu/course/pp530) in the Department of Plant Pathology at N. C. State University. Student enrolled in the class have included graduate students from many departments in CALS, county agents, N. C. Dept. of Agriculture pesticide administrators, N.C. Dept. Envir. 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. 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. Guest lecturers in the past few years have included: JoAnn Burkholder (water quality), Tom Regan, (animal rights), Fred Gould (GM crop plants). This class is now a required part of the graduate curriculum in Plant Pathology at NCSU.
The co-PI (Hu) organized a new class in Soil Ecology that was taught for the first time in the fall of 2001. The other co-PI (Gumpertz) teaches classes in multivariate and spatial statistical techniques. The PI and coPI (Hu) also recently began team-teaching a new class in BT 595E “Ecology, Evolution and Diversity” with a group of faculty from Botany, Microbiology and Plant Pathology. 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.
The PI’s have participated in the Carolina Farm Stewardship Association meetings for the past several years and presented data and posters on the work from this project. A field day was also conducted in the fall of 2001 at Alex Hitt’s farm as a part of the Southern SARE meeting. Data was presented to several groups that toured the farm. Posters have been presented on the work at the last three meetings of the American Phytopathological Society and a poster will be presented in New Orleans at the American Society of Microbiology.
Raw data from the farmer collaborators will be tabulated and given to individual grower collaborators and papers will be submitted to the journal Applied Soil Ecology.
Glenn, D. L. and Ristaino, J. B. 2002. Functional and species composition of soil microbial communities from organic and conventional field soils in North Carolina. Phytopathology 92:S30.
Lui, Bo, and Ristaino, J. B. 2003. Microbial community structure in soils from organic and conventional agroecosystems. Phytopathology 93:S 53.
Lui, Bo, and Ristaino, J. B. 2003. Dispersal of Phytophthora capsici in soils from conventional and organic agro- ecosystems. Phytopathology 93:S53.
Ristaino, J. B. Ecologically-based management of soilborne pathogens. Carolina Farm Stewardship Meeting, Rock Hill, SC, Nov. 8, 2003.
Cong T., Hu, S. and Ristaino, J. B. 2004. Sustaining soil microbial biomass and N supply for crops in a US coastal sandy soil: effects of straw mulching and organic inputs. Agric. Ecosys. Envir. submitted.
Our data clearly indicate that soils from organic farms were more suppressive to Southern blight than those from conventional farms. The actual mechanisms of disease suppression are still under investigation. It may be that specific communities of microorganisms such as thermophiles present in composted organic wastes are suppressing germination of pathogen sclerotia. Soils from organic farms had higher microbial activity. The specific identity of those communities and deliberate introduction of microbes into soils may be possible in the future to change conducive soils to disease suppressive soils. Further experiments are necessary to confirm these results.
We are still in the process of analyzing the data and developing final publications. We intend to present the data at future grower conferences and will share data collected on individual farms with the respective growers. The data will also be shared with extension faculty so that the information can be disseminated to a wider audience including both conventional and organic growers.
Areas needing additional study
Further research will be needed to determine the identity of the bacterial and fungal communities present in soil from the organic, conventional, and sustainable systems. In addition, multivariate analysis of the functional microbial communities in soil is underway. We plan to determine whether functional or species diversity is more important for disease suppression.