NC125: Biological Control of Soil-and Residue-Borne Plant Pathogens

(Multistate Research Project)

Status: Inactive/Terminating

Project Menu

SAES-422 Reports

Annual/Termination Reports:

[02/15/2002] [07/11/2003] [12/28/2002] [05/03/2004] [03/31/2005]

Date of Annual Report: 02/15/2002

Report Information

Annual Meeting Dates: 11/08/2001 - 11/09/2001
Period the Report Covers: 01/01/2001 - 12/01/2001

Participants

Berlin Nelson, North Dakota State University
Gary Yuen, University of Nebraska
Gene Safir, Michigan State University
Charlie Martenson, Iowa State University
Steve Pueppke, Administrative Advisor
Anne Desjardins, USDA-ARS-NCAUR
Daren Eastburn, University of Illinois
Don Kobayashi, Rutgers University
Carol Windels, University of Minnesota
Brian McSpadden Gardener, Ohio State University
Karen Garrett, Kansas State University
Ann Macguidwin, University of Wisconsin

Brief Summary of Minutes

Minutes of the 2000 meeting were approved, with an ensuing discussion concerning minutes, annual reports and committee project proposal. In an effort to better coordinate transfer of information (especially for new representatives), the committee agreed that all members should receive electronic copies of meeting minutes, annual reports and the current project description.

Comments from the Administrative Advisor: Dr. Steve Pueppke discussed that the midterm review for NC-125 will take place this year. The requirements for the formal evaluation can be found at www.wisc.edu/ncra, and will involve a written report with directors. The evaluation will likely take place during the spring of 2002.

The future of agricultural funding sources was discussed. Intellectual properties are becoming of greater importance in agriculture. Establishing the mechanisms for the preservation of intellectual property is a big concern, since it is in conflict with the concept of land grant institutions. The need for new policies and common policies for all institutions is necessary.

Announcements were made stating that Dr. McSpadden Gardener is the new Ohio State University representative and Dr. Ann Macguidwin is the new Wisconsin representative.

The committee took a tour of the USDA facilities associated with biocontrol research. The tour was followed by presentations of biocontrol researchers at USDA. Dr. Pat Slininger presented research on biocontrol of potato dry rot and sprouting. Dr. David Schisler presented research on biocontrol of Fusarium head blight of wheat, Dr. Mark Jackson presented research on producing and stabilizing fungal biopesticides, and Dr. Robert Behle presented research on improving residual activity of biological pesticides.

The committee meeting continued with state reports. Dr. G. Yuen (Nebraska), reported on the status of biocontrol of turfgrass diseases with Lysobacter sp. strain C3. Dr. D. Kobayashi (New Jersey) updated the progress of the molecular characterization of biocontrol traits in Lysobacter sp. strain C3. Dr. A. Macguidwin, (Wisconsin) discussed refining crop loss models for potato early dying for growers in a potato eco-label program. Dr. B. McSpadden Gardener (Ohio) presented information on studying the microbial ecology of agricultural soils as it pertains to biocontrol. Dr. D. Eastburn (Illinois) reported on the characterization of mycoviruses of Fusarium solani f. sp. glycines. Dr. R. Proctor and Dr. A. Desjardins (Illinois) reported on biocontrol of Fusarium verticilliodes using strains that do not produce the mycotoxin, fumonisin. Dr. C. Martinson (Iowa) reported on white mold control with Sporidesmin. Dr. C. Windels (Minnesota) described the use of green cover crop and soil solarization for suppression of Aphanomyces cochlioides. Dr. B. Nelson (North Dakota) reported on biocontrol of Rhizoctonia solani and degradation of sclerotia of Sclerotinia. Dr. K Garrett (Kansas) discussed the use of models to manage wheat planting for escape from take-all. Dr. G. Safir (Michigan) updated progress on expanding simulation models at the regional scale for monitoring disease.

The committee thanked Dr. Desjardins for her service and hosting the 2001 meeting. Dr. B. Nelson suggested that a letter of appreciation for use of the facilities and acknowledging the time and effort of researchers at the facility should be drafted by the new secretary or chair, and sent to the director of USDA-ARS-NCAUR.

Dr. Don Kobayashi, Rutgers University, secretary, will automatically advance to chair. Dr. Brian McSpadden Gardener was elected as the new secretary. The next meeting site was selected as New Brunswick, NJ. The tentative date was agreed upon as some time in Oct/Nov.

Meeting was adjourned at 12:00 pm, November 9, 2001.

Accomplishments

PROGRESS OF THE WORK AND PRINCIPAL ACCOMPLISHMENTS: OBJECTIVE I. Develop systems for effective utilization of microbial amendments for biocontrol of soilborne plant pathogens in the North Central agroecosystem. A wide range of research on this objective by members of the NC-125 Committee was continued in 2001. The abundance and diversity of indigenous biocontrol Pseudomonas species that produce antifungal metabolites were examined in the rhizosphere of tomato and cabbage plants grown in OH. Manure amendments increased the number of pseudomonads of all types, but compost also enriched for producers of the antifungal metabolite pyoluteorin. Host genotypes and growth conditions (greenhouse vs. field) impacted the structure of rhizosphere microbial populations in OH. Disease control of Sclerotinia sclerotiorum on soybean in IA was prolonged for up to five years after soil incorporation of the sclerotial parasite Sporidesmium sclerotivorum. Experiments were continued to optimize inoculum production for S. sclerotivorum, with alginate pellets in SM-4 medium providing the fastest production of spores. In NE, Lysobacter sp. C3, formerly reported as a strain of Stenotrophomonas maltophilia, was shown effective in controlling a broader range of diseases, including wheat head blight, wheat stem rust, and anthracnose in turfgrass. The survival of Lysobacter C3 in turfgrass canopies following exposure to solar ultraviolet was related to canopy density and canopy height. The efficacy of fumonisin gene knock-out mutants in reducing fumonisin production in maize by Fusarium verticillioides was demonstrated in field tests in IL and IA. Thus, fumonisin-nonproducing strains have potential as biological control agents for reducing fumonisins in maize. In KS, a model was developed to determine how different seeding patterns of wheat might affect yield loss to Gaeumannomyces graminis var. tritici when wheat was direct-drilled into fields where wheat was infected with take-all in the previous season. Planting parallel to and in between the previous year=s rows resulted in a much lower predicted loss in yield than angled planting, provided there was not an important offset in the position of the parallel planting. In MI, a model is being developed for assessment of agroecosystems and biological control potential for the North Central Region. A Modeling Application System Integrative Framework has been developed to accommodate large amounts of spatial-temporal outputs from regional scale simulation experiments. In WI, crop loss models for potato early dying are being refined for growers in a potato eco-label program. Objective II. Develop systems to manage the indigenous microflora to suppress diseases caused by soilborne plant pathogens. To better characterize the structure of microbial communities, a high-throughput T-RFLP-based assay of ribosomal DNA sequences (FT-ARDRA) was developed in OH. Significant differences were observed in the bacterial communities colonizing corn and soybean plants grown in the same soil. Abundance and diversity of 2,4-diacetylphloroglucinol (DAPG)-producing biocontrol pseudomonads inhabiting Ohio soils was investigated. Rhizospheres of corn and soybean plants harbored sufficient populations of these biocontrol bacteria to suppress root diseases. RFLP analysis and partial sequencing of selected genes was used to identify different genotypes of DAPG-producing pseudomonands that might differ in adaptability to diverse soil environments and other traits that might affect biocontrol efficacy. In NE, systemic resistance against Bipolaris sorokiniana was induced by application of Lysobacter sp. C3 to roots of tall fescue, but only localized resistance was elicited by application to leaves. Glucanase-deficient mutants of Lysobacter sp. C3 were able to suppress leaf spot in tall fescue and damping-off in sugarbeet, indicating that glucanase activity was not required for biocontrol. Rigorous genetic analysis, and nutritional and biochemical tests in NJ clarified the taxonomy of Lysobacter sp. C3. This biocontrol strain is closely related to another biocontrol agent, strain N4-7, and to bacteria from hydrothermal vents. Chitinases of strain C3 were cloned and characterized, and a type III secretion pathway was identified by homology with genes from Xanthomonas. To study biology of binucleate Rhizoctonia in ND, strains with the carboxin resistance gene were obtained by transformation. The resulting transformants, however, were less efficient biocontrol agents than the wild-type strain, due in part to inefficient colonization and penetration of plant surfaces. Experiments demonstrated that resistance induced in soybean by binucleate Rhizoctonia is localized but is not systemic. Preliminary experiments identified Bacillus spp. isolated from sclerotia of S. sclerotiorum as potential degraders of sclerotia. Survival of oospores of Aphanomyces cochlioides in the soil on sugar beet hypocotyl pieces is being investigated in MN under different field conditions with cover crop residues and soil solarization treatments. Techniques for identification of viable, dead and decomposing oospores have been developed. An ascospore-nonproducing mutant of Gibberella zeae was obtained by deleting the mating locus and was applied to maize stalk pieces to produce an inoculum for field tests. A field test of the mutant strain in IL indicated that ascospore production on maize stalk residues can play a major role in wheat head blight epidemics. The relationship of mycoviruses to virulence of Fusarium solani f. sp. glycines, causal agent of sudden death syndrome of soybeans, is being investigated in IL. Pathogencitiy tests in the greenhouse indicated some significant differences in disease severity between isolate groups with different mycovirus banding patterns. These experiments are being repeated and continued with isolates that have been cured of mycovirus infection by treatment with heat or cycloheximide. USEFULNESS OF FINDINGS: Research in this project has expanded our knowledge of which organisms offer potential as biocontrol agents for commercial development. Basic biological studies continue to give us new insights into how biocontrol works and how it can be utilized to benefit agriculture. New tools are being developed to aid in the understanding of biocontrol and its applications. New factors affecting biocontrol continue to be discovered for pathogen-crop systems. An important contribution of the research is an increase in understanding the limitations of biocontrol and the types of agricultural systems where biocontrol has the most utility. STATEMENT OF ACCOMPLISHMENTS: Significant accomplishments have resulted from this project. Potential biocontrol microorganisms have been identified and tested on various crops and under different agricultural systems. Known biocontrol microbes have been tested on new crops and information on their activities expanded. Factors affecting biocontrol activities were identified and interactions of biocontrol-pathogen-host were studied. Diseases not previously tested for control by biocontrol microbes were studied. The mechanisms of biocontrol were elucidated for certain biocontrol agents, giving us new directions on approaching the problem. New tools for the study and understanding of biocontrol were developed. New strategies for using biocontrol agents were developed and limitations on biocontrol of certain pathogens were discovered. WORK PLANNED FOR NEXT YEAR: The NC-125 project objectives are similar to the past project. Research will continue on many of the same crop systems, but new members will be contributing information on different diseases, new biocontrol organisms and new approaches to biocontrol.

Publications

McGuire, M. R., R. W. Behle, H. N. Goebel, and T. C. Fry. 2000. Calibration of a sunlight simulator for determining solar stability of Bacillus thuringiensis and Anagrapha falcifera nuclear polyhedrovirus. Environ. Entomol. 29:1070-1074. Tamez-Guerra, P., M. R. McGuire, R. W. Behle, J. J. Hamm, R. H. Sumner, and B. S. Shasha. 2000. Sunlight persistence and rainfastness of spray-dried formulations of baculovirus isolated from (Lepidoptera: Noctuidae). J. Econ. Entomol. 93:210-218. Kilic, O., and Yuen, G. Y. 2001. Induced resistance by Stenotrophomonas maltophilia strain C3 suppresses conidial germination and leaf spot development by Bipolaris sorokiniana. Phytopathology 91:S48. Yuen, G.Y., Jochum, C.C., Giesler, L.J., Shulski, M.D., Walter-Shea, E.A., Hubbard, K.G., and Horst, G.L. 2001. UV-B Biodosimetry in Turfgrass Canopies. Crop Science 41: in press. Yuen, G.Y., and Kilic, O. 2001. Evidence of induced resistance in the control of Bipolaris sorokiniana in tall fescue by Stenotrophomonas maltophilia C3. Int. Turfgrass Soc. Res. J. 9:736-741. Yuen, G.Y., and Zhang, Z. 2001. Control of brown patch using the bacterium Stenotrophomonas maltophilia C3 and culture fluid. Int. Turfgrass Soc. Res. J. 9:742-747. Yuen, G.Y., Steadman, J.R., Lindgren, D.T., Schaff, D., and Jochum, C. 2001. Bean rust biological control using bacterial agents. Crop Protection 20:395-402. Zhang, Z., Yuen, G.Y., Sarath, G., and Penheiter, A. 2001. Chitinases from the plant disease biocontrol agent, Stenotrophomonas maltophilia C3. Phytopathology 91:204-211. Kobayashi, D.Y., Reedy, R.M., Bick, J.A., and Oudemans, P.V. Characterization of a chitinase gene from Stenotrophomonas maltophilia and its involvement in biological control. Appl. Environ. Microbiol.: accepted with revisions (9/01). Bick, J.A., Reedy, R.M., and Kobayashi, D.Y. 2001. Genetic analysis of chitinolytic activity from the biocontrol agent Stenotrophomonas maltophilia strain C3. Phytopathology 91:S8-9. Reedy, R.M., and Kobayashi, D.Y. 2001. Evidence for a type III secretory pathway in the biocontrol bacterium Stenotrophomonas maltophilia strain C3. Phytopathology 91:S75. McSpadden Gardener, B., and Weller, D.M. 2001. Changes in populations of rhizosphere bacteria associated with take-all disease of wheat. Appl. Environ. Microbiol. 67: 4414-4425. McSpadden Gardener, B., et al. 2001. A rapid polymerase chain reaction-based assay characterizing rhizosphere populations of 2,4-diacetylphloroglucinol-producing bacteria. Phytopathology 91:44-54. Marvelli, R. A., Eastburn D. M., and Domier, L. 2001. Detection of mycoviruses in Fusarium solani f. sp. glycenes. Phytopathology 91:S58. Dyer, A.T., and Windels, C.E. 2001. Visual viability assay for oospores of Aphanomyces cochlioides. Phytopathology 91:S25. Dyer, A.T., and Windels, C.E. 2001. Methods for assessing viability of Aphanomyces cochlioides oospores. J. Sugar Beet Res. 38:65. Dyer, A.T., and Windels, C.E. 2001. Methods for assessing viability and maturation of Aphanomyces cochlioides oospores. 2000 Minnesota-North Dakota Sugarbeet Res. Ext. Rept. 31:243-253. Nelson, B D., Christianson, T., and McClean, P. 2001. Effects of bacteria on sclerotia of Sclerotinia sclerotiorum. Proceedings of the IV International Sclerotinia Conference. Central Science Laboratory. York, England. July 2001. Pages 39-41. Nelson, B. D. 2001. Adaptation and importance of Sclerotinia sclerotiorum in North Dakota. Proceedings of the IV International Sclerotinia Conference. Central Science Laboratory. York, England. July 2001. Pages 177-178. Nelson, B., and Christianson, T. 2001. Biology of Binucleate Rhizoctonia. P. 48-49. In Proceedings Biocontrol in a New Millennium: Building for the Future on Past Experience. D. M. Huber, editor. Estes Park, CO. October 26-29, 2000. Nelson, B., Helms, T., Christianson, T., Martin, G., Harrington, G., and Mars, G. 2000. Evaluation of soybean cultivars for resistance to Sclerotinia stem rot, 1999. Biol. Cult. Test Cont. Plant Dis. 16:80. Mars, G. A., Nelson, B. D., and Helms, T. C. 2001. Effect of Sclerotinia stem rot on seed yield of two soybean cultivars inoculated at two growth stages. Phytopathology 91: Nelson, B. and Lamey, A. 2000. Sclerotinia diseases of sunflower. Cooperative Extension Service Circular PP-840. March 2000. North Dakota State University. 8 pp.

Impact Statements

Date of Annual Report: 07/11/2003

Report Information

Annual Meeting Dates: 10/21/2002 - 10/22/2002
Period the Report Covers: 01/01/2002 - 12/01/2002

Participants

Steve Pueppke, Administrative Advisor
Phil Colbaugh, University of Texas
Darin Eastburn, University of Illinois
Don Huber, Purdue University
Andreas Westphal, Purdue University
Don Kobayashi, Rutgers University
Berlin Nelson, North Dakota State University
Gene Safir, Michigan State University
Gary Yuen, University of Nebraska
Gwen Beattie, Iowa State University

Brief Summary of Minutes

The meeting was called to order by Don Kobayashi at 10:00 a.m.

Agenda:

Introduction of attending members

Approval of agenda and minutes from 2001 meeting in IL.

Election of new officers

Comments by Administrative Advisor

Old Business

New Business

State Reports

The agenda and minutes of 2001 were approved, with the addition that Karen Garrett (Kansas) was a new member in 2001. Darin Eastburn was elected secretary for 2003, and will be chair the following year. Brain McSpadden-Gardner will be chair in 2003. Next year‘s meeting place will be decided by the new chair.

Steve Pueppke mentioned the process of approving a new project which must be submitted by October of 2003. If all goes a planned it would be approved around July 2004. Steve discussed the format and process of putting a new project together.

Steve mentioned that any new members of the committee will need to have their experiment station director notify the administrative advisor about the change. The Hatch budget was discussed. The NRI budget is expected to be increased next year. IFAFS is not expected to survive this next year. Steve also discussed issues of agrosecurity.

There was a long discussion about terrorism and plant diseases. The committee had a discussion on how these issues relate to our next project write up.

Members who are listed but do not attend should be asked whether they really want to be membrs. It looks bad to the directors when there are people listed who never attend. Luis Del Rio will probably be representative from North Dakota Next year.

There was a long discussion of the new project, and the writing team was identified. State reports were then given. The regular business meeting adjourned at 6:30 p.m.

Accomplishments

Effects of in-furrow micronutrient applications on soybean sudden death syndrome have been identified and are being developed for control. An integrated software program called MASIF has been developed with the leadership of Michigan State University and is being used to model losses due to plant disease. The mechanisms by which Lysobacter enyzmogenes controls foliar diseases of grasses are being determined, so as to provide a rationale for use of this organism in biocontrol. This information is being combined with molecular analysis of specific genes that function in biocontrol.

Publications

Impact Statements

Date of Annual Report: 12/28/2002

Report Information

Annual Meeting Dates: 12/28/2002 - 12/28/2002
Period the Report Covers: 12/28/2002 - 12/28/2002

Participants

Brief Summary of Minutes

Accomplishments

Publications

Impact Statements

Date of Annual Report: 05/03/2004

Report Information

Annual Meeting Dates: 10/23/2003 - 10/24/2003
Period the Report Covers: 01/01/2003 - 12/01/2003

Participants

Participants: G. Beattie (IA); L. Del Rio (ND); D. Eastburn (IL); K. Garrett (KS); D. Huber (IN); D. Kobayashi (NJ); B. MacSpadden Gardner (OH); G. Safir (MI); A. Westphal (IN); G. Yuen (NE); Administrative Advisor: S. G. Pueppke (IL)

Brief Summary of Minutes

Committee membership: A. Westphal was nominated as committee secretary and agreed to act as chair in 2004. Committee members reintroduced themselves and briefly described their research expertise and committee related activities. Written state reports were passed out by several attendees.

New project proposal for NC-125: The committee focused most of its time on the development of the renewal project. S. Pueppke discussed the history of the committee, funding allocations, and the nature of collaborations under the MSRP structure. B. McSpadden Gardener presented an overview of the renewal process including a timeline for submission, the review process, and related information described by materials available on the NIMSS web site. Discussion ensued as to the draft version of the renewal project prepared by the writing committee. Suggestions for additions and revisions followed. Particular emphasis was put on developing collaborations and planning extension-related activites. D. Eastburn led the writing activities that led to a nearly complete revision by the end of the meeting. Additional refinements were made by the writing committee following the meeting and the final renewal proposal was submitted for review to the entire committee before submission.

Accomplishments

Three commercially produced biological seed treatments were evaluated for their effects on the development of sudden death syndrome of soybeans (SDS), caused by Fusarium solani f. sp. glycines, in an inoculated field setting. Seed of a soybean variety with known susceptibility to SDS was treated with formulations of Yield Shield WP (Bacillus pumilus GB34, 0.1 oz per 100 lbs seed), Bio Yield F (Bacillus amyloliquefaciens GB 99, and Bacillus subtilis GB 122, 0.5 fl. oz. per 100 lbs seed), Kodiak F (Bacillus subtilis GBO3, 0.5 fl. oz. per 100 lbs seed) prior to planting. Treated seed, as well as non-treated control seed, were planted in 25 ft. rows with 30 inch row spacing into plots that were inoculated or non-inoculated with Fusarium solani f. sp. glycines. Inoculum was applied as colonized sorghum seed at a rate of 300 lbs/acre, using a seed drill to a depth of six inches seven weeks prior to planting. Plots were monitored for foliar symptoms of SDS throughout the season, and yield data were recorded for each plot. Only very mild foliar symptoms were observed on a few plants in late August. Most of the plants showed no foliar symptoms during the season. Analysis of yield data showed no significant differences between any of the seed or inoculation treatments. Hot weather conditions at the beginning of August may have disrupted the disease development process. Very little SDS was observed in Illinois this season. We have studied the effect of crop sequence and tillage, over time, on Mn transition states in soil as this might affect biological control activity and predispose plants to disease. There were interactions between tillage (Fall Chisel and No Till) and Crop rotation/sequence (continuous corn, continuous soybean, corn-soybean-wheat rotation in various combinations). Manganese availability was highest in the continuous corn rotation and lowest in the continuous soybean rotation which might explain the limited practice of continuous soybean compared to continuous corn. Manganese was not as available with crop rotations of corn and soybean with wheat as with continuous corn, but better than continuous soybeans (Table 1). The order of highest Mn availability was continuous corn > corn in a corn-soybean-wheat sequence > soybeans in a corn-soybean-wheat rotation > wheat in a corn-soybean-wheat sequence > continuous soybean Manganese was generally more available in a Fall chisel than no-till system. Since some soilborne plant pathogens oxidize Mn+2 to the non-available oxidized Mn+4 form, effects of the crop on Mn valence states in the soil could predispose to disease and enhance pathogen virulence. Many proposed biocontrol agents are Mn reducers and could off-set the effects of reduced availability induced by the pathogen or crop rhizosphere effects. The basis for increased take-all of wheat commonly observed after glyphosate burn-down herbicide treatments or following glyphosate-resistant crops was evaluated on a low and sufficient Mn soil.The increase in take-all following glyphosate-resistant soybeans indicates that the effect is indirect through soil microbial interactions influenced by root exudates or residue decomposition. Presence of the glyphosate-resistant gene in corn and soybeans quite specifically reduced the uptake efficiency of Mn (tissue concentrations). Application of glyphosate further reduced Mn uptake and utilization efficiency. Manganese applied + or ? four days around the glyphosate ?event? was not translocated to new tissue. Initial data suggests that application 8-10 days after glyphosate may avoid the glyphosate-immobilization and reduced physiological efficiency. Tank-mixing the micronutrients (Cu, Mn, Zn) as well as P and Ca not only reduces their uptake by plants, but also reduces the herbicidal efficacy of the glyphosate. Roundup formulation influenced the effect on micronutrient efficacy with the weathermax formulation having less interaction than the Roundup Ultramax formulation. Amplification of phylogenetically significant sequences (i.e. ribosomal and actin genes) was attempted using a mix of previously described and newly designed oligonucleotide primers. The complexity of soil and rhizosphere DNA used as template precluded clean amplification of DNA fragments of the expected size for several of the tested primer sets. Nonetheless, positive results were obtained for previously published primers targeting the ribosomal sequences of Archaea, Fungi, Bacteria, and Pseudomonas spp., as well as the nitrogenase gene, nifH, from rhizobia. Success was also achieved with primers designed in our laboratory targeting the ribosomal genes of Bacillus, Burkholderia, Trichoderma, and the actin genes of Pythium and Phytophthora spp. The phylogenetic origin of amplified sequences was determined by cloning and sequencing multiple products from each reaction set. These results provided us with a significant improvement in our ability to characterize the abundance and diversity of the many different microbial taxa inhabiting agricultural soils. We used these primer sets to characterize microbial diversity across different environments. DNA was isolated from soils and rhizospheres sampled at multiple sites throughout the state of Ohio. Microbial community profiles were generated that allowed for comparisons between crops, sites, and treatments. Microbial community structure was found to differed dramatically with microhabitat (e.g. soil vs rhizosphere), but also with crop (corn vs. soybeans). In contrast, fewer differences were observed between sites despite significant variation in soil types and prevailing weather conditions. These results indicate the relative importance of plant species in determining the active fraction of soil microbial communities. Such species-dependent variation indicates: i) A comprehensive evaluation of microbiotic contributions to plant health across large geographic areas can be achieved. ii) The relative contributions of different microbial populations to plant health can be calculated using a combination of community profile and disease assessment data. iii)) Crop selection is a major controlling factor for microbial processes at the field scale. We have completed two projects that have quantified the influences of biocontrol agents on rhizosphere bacterial population structure in two different collaborative efforts. In the first, we examined the effects of a wild-type DAPG producer and a near isogenic strain that was genetically modified to produce phenazine-1-carboxylic acid (PCA). In the second, we examined the influences of Trichoderma harzianum T382 as compared to the composition of various potting mixes. In both instances, colonization of the rhizosphere by the biocontrol agents was verified. Using T-RFLP analyses, we determined that multiple shifts in bacterial populations occurred following applications of the different biocontrol agents. However, the magnitude of such changes was generally small and the effects were inconsistent over time in independent replicates. These data indicate that application of these biocontrol agents is, like other farming practices, a disturbance, but one with no consistent impact on the phylogenetic composition of root colonizing bacterial populations. These data also indicate that there is a high degree of metabolic redundancy among phylogenetically distinct bacterial populations. IIB. Elucidate biochemical and genetic basis for the interactions. (NE) Efforts to further characterize metabolites produced by L. enzymogenes C3 that might be involved in biological yielded factors that have positive and negative effects on biocontrol efficacy. A small molecular weight, heat-stable antifungal factor, presumably involving a family of lipopeptide antibiotics, was found to cause abnormal hyphal growth in which hyphal polarity was disrupted. The factor was effective in vitro against all plant pathogenic fungi tested, as well as oomycetes. Using a mutant strain of C3 (7A-4) with reduced antibiotic excretion, evidence was found that the antifungal factor might have a role in biocontrol. When cell-free fluid from a broth culture of 7A-4 was applied to tall fescue foliage, it was less effective in suppressing Bipolaris leaf spot than fluid from a culture of the wild type strain. C3 also produces a phytotoxic factor when grown in vitro or when applied to seed. The factor inhibits seed germination and reduces root growth in all graminaceous plants tested; some dicots (soybeans, tobacco) are sensitive to the factor, but sugarbeet is not. While the factor does not affect plants when it is applied to foliage, its effect on root growth is a concern when C3 is applied as a seed or root treatment.

Publications

Blouin-Bankhead, S., Landa, B.B., Lutton, E., Weller, D.M., McSpadden Gardener, B. 2003. Minimal changes in rhizobacterial population structure following root colonization by wild type and transgenic biocontrol strains. FEMS Microbiol Ecol. submitted 12/18/03. Del Rio, L.E., R. Henson, and T. Gulya. 2003. Use of Intercept for control of Sclerotinia on dry beans, canola, and sunflower in North Dakota. Proc. 2003 Sclerotinia Initiative Annual Meeting. Bloomington, MN 23pp. Garrett, K.A., M. Kabbage, and W. W. Bockus. 2003. Managing for fine-scale differences in inoculum load: seeding patterns to minimize loss to Gaeumannomyces graminis var. tritici. Precision Agriculture in press Huber, D.M. 2003. Nutrient-Disease interactions in the take-all disease of cereals. Proc. Symposium on Take-all, April 2003, Temuco, Chili Huber, D.M. 2003. The role of plant nutrition in disease. Proc. Symposium March 2003, Relacao entre Nutricao de Plantas e Incidencia de Doencas, Piracicaba, SP, Brazil. Kilic-Ekici, O. and Yuen, G.Y. 2003. Induced resistance as a mechanism of biological control by Lysobacter enzymogenes strain C3. Phytopathology 93:1103-1110. Mathew, F.M. and L.E. del Rio. 2003. Development of a less complex medium for production of Sporidesmium sclerotivorum. On-line publication no. P-2004-0030-NCA. www.apsnet.org/meetings/div/nc03abs.asp McSpadden Gardener, B. 2003. Ecology of Bacillus species in agricultural soils. Phytopathology 93:S117. Abstract. McSpadden Gardener, B. 2003. Microbial biodiversity in agricultural systems: General and specific barriers to inoculation. Phytopathology 93:S100. Abstract. McSpadden Gardener, B. R. Joshi, L. Gutierrez, E. Lutton, and S. Grewal. 2003. Options and challenges for assessing microbial diversity in the phyllosphere. Phytopathology 92:S106. Abstract. Ortiz, O., K. A. Garrett, J. J. Heath, R. Orrego, and R. J. Nelson. 2003. Management of potato late blight in the Andean highlands: Evaluating the benefits of Farmer Participatory Research and Farmer Field Schools. Plant Disease. in press Palumbo, J.D., Sullivan, R. and Kobayashi, D.Y. 2003. Molecular characterization and expression in Escherichia coli of three ?-1,3-glucanase genes from Lysobacter enzymogenes strain N4-7. J. Bacteriol.: 185:4362-4370. Reedy, R.M. and Kobayashi, D.Y. 2003. Evidence for induction of the type III secretory pathway in the biocontrol bacterium Lysobacter enzymogenes strain C3 in the presence of fungal cell constituents. Phytopathology 93: S73. Sabaratnam, S. and G. A. Beattie. 2003. Differences between Pseudomonas syringae pv. syringae B728a and Pantoea agglomerans BRT98 in epiphytic versus endophytic colonization of leaves. Applied and Environmental Microbiology 69:1220-1228. Sullivan, R.F., Holtman, M.A., Zylstra, G.J., White, J.F. and Kobayashi, D.Y. 2003. Identification of Two Biological Control Agents for Plant Diseases as Lysobacter enzymogenes based on phylogenetic analysis of 16S rDNA, fatty acid composition and phenotypic characteristics. J. Appl. Microbiol. 94: 1079-1086.

Impact Statements

Growers (particularly in NE, MN, KS, and IN) have new management strategies that are based on research done by the committee

Date of Annual Report: 03/31/2005

Report Information

Annual Meeting Dates: 08/04/2004 - 08/04/2004
Period the Report Covers: 01/01/1999 - 12/01/2004

Participants

* G. Beattie (IA)
* L. Del Rio (ND)
* D. Eastburn (IL)
* K. Garrett (KS)
* D. Huber (IN)
* D. Kobayashi (NJ)
* B. McSpadden Gardner (OH)
* G. Safir (MI)
* A. Westphal (IN)
* G. Yuen (NE)
* Administrative Advisor: S. G. Pueppke (IL)

Brief Summary of Minutes

The last meeting of the entire committee was held in Wooster, OH on month/date of 20034. A. Westphal was nominated as committee secretary and agreed to act as chair in 2004. Committee members reintroduced themselves and briefly described their research expertise and committee related activities. Written state reports were passed to the group by several attendees.

New project proposal for NC-125: The committee focused most of its time on the development of the renewal project. S. Pueppke discussed the history of the committee, funding allocations, and the nature of collaborations under the MSRP structure. B. McSpadden Gardener presented an overview of the renewal process including a timeline for submission, the review process, and related information described by materials available on the NIMSS web site. Discussion ensued as to the draft version of the renewal project prepared by the writing committee. Suggestions for additions and revisions followed. Particular emphasis was put on developing collaborations and planning extension-related activities. D. Eastburn led the writing activities that led to a nearly complete revision by the end of the meeting. The writing committee applied refinements following the meeting and review by the entire committee. The final renewal proposal was submitted. Renewal was not granted. A subgroup of the committee met at the APS meetings at Charlotte, NC in 2004.

Accomplishments

Objective I. Compare efficacy of biocontrol systems on economic plants in the North Central Region. Objective IA. Compare methods of production, delivery and enhancement of applied and indigenous biocontrol agents. Three commercially produced biological seed treatments were evaluated for their effects on the development of sudden death syndrome of soybeans (SDS), caused by Fusarium solani f. sp. glycines, in an inoculated field setting. Seed of a soybean variety with known susceptibility to SDS was treated with formulations of Yield Shield WP (Bacillus pumilus GB34, 0.1 oz per 100 lbs seed), Bio Yield F (Bacillus amyloliquefaciens GB 99, and Bacillus subtilis GB 122, 0.5 fl. oz. per 100 lbs seed), Kodiak F (Bacillus subtilis GBO3, 0.5 fl. oz. per 100 lbs seed) prior to planting. Treated seed, as well as non-treated control seed, were planted in 25 ft. rows with 30 inch row spacing into plots that were inoculated or non-inoculated with Fusarium solani f. sp. glycines. Inoculum was applied as colonized sorghum seed at a rate of 300 lbs/acre, using a seed drill to a depth of six inches seven weeks prior to planting. Plots were monitored for foliar symptoms of SDS throughout the season, and yield data were recorded for each plot. Only very mild foliar symptoms were observed on a few plants in late August. Most of the plants showed no foliar symptoms during the season. Analysis of yield data showed no significant differences between any of the seed or inoculation treatments. Hot weather conditions at the beginning of August may have disrupted the disease development process. Very little SDS was observed in Illinois this season. We determined if tomato transplants treated with BioYield" or DAPG-producing bacteria native to Ohio soils have the ability to promote plant growth or induce host resistance. The bacteria tested were present in a commercialized biological product or rhizobacteria isolated previously from tomato or soybean rhizosphere. Bacteria treatment of tomato seeds did not alter germination. However, all three of the bacteria treatments significantly decreased the root-to-shoot ratio of treated plants compared to water treated plants. The bacteria treatments resulted in plants with greater shoot tissue than those treated with water. Therefore, we concluded that all three of these bacteria treatments were capable of promoting plant growth. This is a novel finding that DAPG-producing P. fluorescens strains from tomato and soybean rhizosphere are capable of promoting plant growth in the absence of root disease pressure. The enzyme activity in green tissue from these treated seedlings was not significantly different from plants treated with water. This is an indication that these bacteria treatments do not induce defense related enzyme activity. It may be interesting to further investigate the ability of DAPG-producing P. fluorescens strains to promote plant growth of transplant tomatoes and other vegetables. In greenhouse studies, Trichoderma sp and mycorrhizal inoculation protected plants from infection by Fusarium oxysporum. Field results in 1999-2000 suggested that both formononetin and arbuscular mycorrhizal inoculation can increase plant height of asparagus and reduce Fusarium root lesions. A cooperative effort with S. Gage, Director of the Computational Ecology and Visualization Laboratory at MSU was initiated in 2001 to determine the effects of environment and biocontrol organisms at larger than field scales. A modeling applications systems integrative framework (MASIF) was developed to expand crop models (corn and soybean) from field to multicounty and regional use. This framework allows information derived from crop models to be shown in visual form and links an array of existing visualization, data management, and analytical software to manage the large volumes of model inputs and outputs as well as model execution. The MASIF framework also allows the incorporation of environmental databases, independent of the use of crop models, for biocontrol options at expanded scales. We have studied the effect of crop sequence and tillage, over time, on Mn transition states in soil as this might affect biological control activity and predispose plants to disease. There were interactions between tillage (Fall Chisel and No Till) and Crop rotation/sequence (continuous corn, continuous soybean, corn-soybean-wheat rotation in various combinations). Manganese availability was highest in the continuous corn rotation and lowest in the continuous soybean rotation, which might explain the limited practice of continuous soybean compared to continuous corn. Manganese was not as available with crop rotations of corn and soybean with wheat as with continuous corn, but better than continuous soybeans (Table 1). The order of highest Mn availability was continuous corn > corn in a corn-soybean-wheat sequence > soybeans in a corn-soybean-wheat rotation > wheat in a corn-soybean-wheat sequence > continuous soybean Manganese was generally more available in a Fall chisel than no-till system. Since some soilborne plant pathogens oxidize Mn+2 to the non-available oxidized Mn+4 form, effects of the crop on Mn valence states in the soil could predispose to disease and enhance pathogen virulence. Many proposed biocontrol agents are Mn reducers and could off-set the effects of reduced availability induced by the pathogen or crop rhizosphere effects. The basis for increased take-all of wheat commonly observed after glyphosate burn-down herbicide treatments or following glyphosate-resistant crops was evaluated on a low and sufficient Mn soil. The increase in take-all following glyphosate-resistant soybeans indicates that the effect is indirect through soil microbial interactions influenced by root exudates or residue decomposition. Presence of the glyphosate-resistant gene in corn and soybeans quite specifically reduced the uptake efficiency of Mn (tissue concentrations). Application of glyphosate further reduced Mn uptake and utilization efficiency. Manganese applied + or ? four days around the glyphosate ?event? was not translocated to new tissue. Initial data suggests that application 8-10 days after glyphosate may avoid the glyphosate-immobilization and reduced physiological efficiency. Tank-mixing the micronutrients (Cu, Mn, Zn) as well as P and Ca not only reduces their uptake by plants, but also reduces the herbicidal efficacy of the glyphosate. Roundup formulation influenced the effect on micronutrient efficacy with the weathermax formulation having less interaction than the Roundup Ultramax formulation. Amplification of phylogenetically significant sequences (i.e. ribosomal and actin genes) was attempted using a mix of previously described and newly designed oligonucleotide primers. The complexity of soil and rhizosphere DNA used as template precluded clean amplification of DNA fragments of the expected size for several of the tested primer sets. Nonetheless, positive results were obtained for previously published primers targeting the ribosomal sequences of Archaea, Fungi, Bacteria, and Pseudomonas spp., as well as the nitrogenase gene, nifH, from rhizobia. Success was also achieved with primers designed in our laboratory targeting the ribosomal genes of Bacillus, Burkholderia, Trichoderma, and the actin genes of Pythium and Phytophthora spp. The phylogenetic origin of amplified sequences was determined by cloning and sequencing multiple products from each reaction set. These results provided us with a significant improvement in our ability to characterize the abundance and diversity of the many different microbial taxa inhabiting agricultural soils. We used these primer sets to characterize microbial diversity across different environments. DNA was isolated from soils and rhizospheres sampled at multiple sites throughout the state of Ohio. Microbial community profiles were generated that allowed for comparisons between crops, sites, and treatments. Microbial community structure was found to differed dramatically with microhabitat (e.g. soil vs rhizosphere), but also with crop (corn vs. soybeans). In contrast, fewer differences were observed between sites despite significant variation in soil types and prevailing weather conditions. These results indicate the relative importance of plant species in determining the active fraction of soil microbial communities. Such species-dependent variation indicates: (i) A comprehensive evaluation of microbiotic contributions to plant health across large geographic areas can be achieved. (ii) The relative contributions of different microbial populations to plant health can be calculated using a combination of community profile and disease assessment data. (iii) Crop selection is a major controlling factor for microbial processes at the field scale. We have completed two projects that have quantified the influences of biocontrol agents in rhizosphere bacterial population structure in two different collaborative efforts. In the first, we examined the effects of a wild-type DAPG producer and a near isogenic strain that was genetically modified to produce phenazine-1-carboxylic acid (PCA). In the second, we examined the influences of Trichoderma harzianum T382 as compared to the composition of various potting mixes. In both instances, colonization of the rhizosphere by the biocontrol agents was verified. Using T-RFLP analyses, we determined that shifts in bacterial populations occurred following applications of the different biocontrol agents. However, the magnitude of such changes was generally small and the effects were inconsistent over time in independent replicates. These data indicate that application of these biocontrol agents is, like other farming practices, a disturbance, but one with no consistent impact on the phylogenetic composition of root colonizing bacterial populations. We further determined the abundance of DAPG-producing pseudomonads, native biocontrol populations with the capacity to inhibit the growth of diverse root pathogens. This past year, we evaluated the effects of rotation and compost addition in additional controlled experiments. DAPG producers were found to be most abundant in compost amended plots as compared to non-amended plots, indicating that applied organic matter can stimulate their populations and biocontrol activities. We also observed that the soil conditions altered by rotations significantly affected rhizosphere colonization. These data also indicate that there is a high degree of metabolic redundancy among hylogenetically distinct bacterial populations. These results further indicate that the abundance of native DAPG-producing pseudomonad populations can be managed to some degree. We have characterized the susceptibility of perennial grain accessions to the important diseases tan spot, take-all, barley yellow dwarf, and wheat streak mosaic. We have characterized the differences in timing requirements for eradication of volunteer wheat harboring wheat streak mosaic virus vectors as a function of the method of eradication. We have demonstrated that the tan spot pathogen is more competitive than the leaf rust pathogen, to the extent that the presence of the leaf rust pathogen can facilitate greater tan spot infection under some circumstances. We have described the likely challenges due to disease in perennial grain systems. We have demonstrated that cultivar mixtures can reduce disease for both tan spot and leaf rust, though the effect is greater for leaf rust as predicted by the life history characteristics of the leaf rust pathogen. We have proposed a novel method for managing take-all through simple changes in planting patterns that we predict can have a substantial impact to reduce yield losses. In the study of plant disease ecology of tallgrass prairie systems, we have determined that seven dominant tallgrass prairie species are slightly susceptible to tan spot pathogens from wheat and three of seven are highly susceptible to take-all pathogens from wheat. We have demonstrated that a common rust pathogen of the tallgrass prairie dominant big bluestem is more abundant in midslope positions. We have shown that three dominant tallgrass prairie species are commonly infected with barley yellow dwarf virus, though the dominant strains are not the same as in Kansas wheat. The non-target effects of Dutch Trig, a fungal biocontrol agent used to protect elm trees against Dutch elm disease were evaluated in a greenhouse based experiment. Dutch Trig is a Verticillium sp. originally isolated from potato. We have been evaluating the pathogenicity of the Dutch Trig fungus on a number of vegetable crops to determine if this biocontrol agent is pathogenic on commonly grown vegetable species known to be susceptible to Verticillium wilt. Greenhouse inoculations with the Dutch Trig organism resulted in typical Verticillium wilt symptoms on tomatoes and eggplant. However the symptoms on Dutch Trig inoculated plants were not as severe as those on plants inoculated with a virulent isolate of Verticillium dahliae isolated from pepper. Peppers inoculated with the Dutch Trig organism were stunted compared to the non-inoculated control plants. Some mild leaf vein necrosis was observed on inoculated broccoli, but no symptoms were observed on horseradish plants inoculated with the Dutch Trig organism. The biocontrol agent Sporodesmium sclerotivorum is a mycoparasite of sclerotia. Infestation of S. sclerotivorum into research plots on several Iowa farms reduced the incidence of Sclerotinia stem rot by an average of 73% in 2000. Biocontrol efficacy was not strongly influenced by the inoculum dosage, i.e., by the density at which the fields were infested with S. sclerotivorum macroconidia. Furthermore, the antagonist was shown to control disease under no-till conditions as well as following incorporation into soil. The effectiveness of S. sclerotivorum at controlling Sclerotinia wilt in sunflower in North Dakota was examined, and although little disease occurred, the fields were found to have been heavily infested with natural S. sclerotivorum strains prior to introduction of the biocontrol strain. The optimal method for mass production of S. sclerotivorum inoculum was found to be the preparation of alginate pellets, with S. sclerotivorum mycelia either inside or outside the pellets. Objective I B. Determine the influences of host and environment on biocontrol efficacy A four-year, multidisciplinary research effort to study the biological effects of transitioning from conventional cash grain production to an organic production system at the University of Illinois research farm continued in the 2004 growing season. Plots of three organic production systems (pasture, cash grains, and vegetable crops) were established on a six acre field site that has been designated for research on organic agriculture. Three amendment treatments (no amendment, manure amendment, and composted plant material amendment) were established as sub-plots within each of the production system plots, applied in the Spring of 2004. In 2004 the cash grain plots were planted to wheat, and the vegetable crop plots were planted to cabbage and broccoli. Soil samples were taken during the growing season, and they will be evaluated for differences in disease suppressiveness to three pathogens, Rhizoctonia solani, Fusarium solani f. sp. glycines, and Phytophthora sojae in greenhouse assays this winter. Soil samples were taken from all plots this summer to establish baseline disease suppression levels. We examined the effects of tillage on soil suppressiveness against Heterodera glycines and conducted a survey for soil suppressiveness against this pathogen in soybean production systems. Further studies on the dynamics of native pathogen antagonists were performed in prairie and agricultural soils. Specifically, comprehensive analyses of the genetic and phenotypic diversity of streptomycetes were continued, focusing on 16S sequences, rep-PCR fingerprints, nutrient utilization, and antibiotic inhibition and resistance profiles. Hypotheses of local adaptation and fitness tradeoffs were tested for native prairie streptomycete communities. Studies of the impacts of green manures on disease intensity on newly established alfalfa stands were established in field plots at Becker, MN and in St. Paul, MN. A major factor that limits biocontrol of foliar diseases is often the poor ability of the antagonists to establish and maintain populations following inoculation onto leaves. Studies addressing the factors influencing bacterial leaf colonization used the species Pantoea agglomerans (previously Erwinia herbicola), which is a species that includes several strains with antagonistic activity against phytopathogens. Bacterial adherence to leaf surfaces was shown to be advantageous to leaf colonization, and P. agglomerans was strongly superior to at least two phytopathogens in the proportion of the population that adhered to leaves. P. agglomerans was found to localize exclusively to leaf surface sites, rather than to endophytic sites like the phytopathogen examined, and thus as a biocontrol agent, P. agglomerans must have mechanisms for tolerating the fluctuating environmental conditions characteristic of leaf surfaces. Although studies with whole-cell water deprivation-responsive biosensors indicated that P. agglomerans cells were not strongly deprived of water on leaves under controlled conditions, P. agglomerans was more tolerant to water deprivation than two phytopathogenic bacterial species. These results suggested that under field conditions, where the fluctuations in water availability on leaves are particularly pronounced, water deprivation tolerance of P. agglomerans may be one factor required for successful population development on leaves. Objective II. Determine mechanisms of action of North Central region biocontrol systems. Objective IIA. Determine the nature of biocontrol agent-crop-pathogen association. Further work was conducted using the phylogenetic markers developed in our laboratory. The complexity of soil and rhizosphere DNA used as template resulted in a mixture of amplification products of diverse phylogenetic origin. This was particularly apparent with the genus-specific probes when relatively few cycles were used in the amplifications. These results, while representing a significant improvement in our ability to characterize the abundance and diversity of the many different microbial taxa inhabiting agricultural soils, indicate that subdominant microbial populations are often obscured in such culture-independent assays. Overall, our results indicate that: (i) A comprehensive evaluation of microbiotic contributions to plant health across large geographic areas will be limited to the numerically dominant populations. (ii) The relative contributions of different microbial populations to plant health can be calculated using a combination of community profile and disease assessment data. (iii) Crop selection is the single most important major controlling factor for microbial processes in the rhizosphere. Evaluation of the biocontrols for the induction of host resistance in vegetables: We set out to determine if Actigard" can induce host resistance in fruit by foliar application. Four and ten days after treatment, fruit tissue from plants treated with Actigard tended to have lower enzyme activity than those of control plants. To contrast, leaf tissue from plants treated with Actigard tended to have higher enzyme activity than the control plants. This is an indication that Actigard treatment induces defense-related enzymes in green tissue but not in fruit tissue. The plants in this investigation were heat stressed for at least 24 hours. This stress resulted in the inability of the experiment to be fully replicated, so, the results are not entirely conclusive. However, the activation of defense related enzymes in green tissue indicates the ability of Actigard treatment to induce host defenses in tomato leaves. It would be interesting to investigate the ability of Actigard treatment to reduce disease severity caused by foliar fungal pathogens common in Ohio. It has already been illustrated that this product is capable of reducing disease severity caused by bacterial diseases of tomato. Mycoviruses, viruses that infect fungi, have been studied in several plant pathogen systems, and in some cases they have been found to affect the pathogens ability to cause disease. We are currently studying the presence of mycoviruses in Fusarium solani f. sp. glycines, causal agent of sudden death syndrome of soybeans, and evaluating their effects on the pathogens ability to cause disease. In other disease systems mycoviruses have been used to develop biological control systems based on the use of hypovirulent isolates. The presence and effects of mycoviruses also may provide insight into a source of variation in characteristics within a pathogen population, giving us more information on basic pathogen biology. Additional virulence assays and reanalysis of data from previous experiments have demonstrated that FSG isolates containing dsRNA mycoviruses are generally less virulent than are isolates which are free of mycovirus infection, although the presence of mycoviruses is not the only factor affecting virulence. Assays comparing virulence levels of mycovirus cured strains with those of their infected parental strains indicate an increased level of virulence in isolates that have been cured of the 10 to 11 kb dsRNA fragments. Based on initial evidence, the 10 to 11 kb dsRNA fragments are believed to be mycoviruses in the Hypoviride group of viruses. Efforts to sequence the dsRNA fragments and further characterize the viruses continue. We have reviewed a number of statistical advances that can be applied to improve plant pathology research. We have introduced meta-analysis to plant pathologists with an example of application in comparative studies of yield loss to disease. IIB. Elucidate biochemical and genetic basis for the interactions. This research has established that manganese oxidation is a virulence factor for the wheat take-all pathogen, Gaeumannomyces graminis, and other plant pathogens. Since manganese is physiologically available to the plant for defense reactions (shikimate pathway) only in the reduced form, oxidation of manganese by the pathogen in the infection court reduces the plants ability to respond to infection. Manganese oxidation occurs through a pathogen produced extracellular enzyme(s) similar to manganese peroxidase. Some pathogen-produced laccases also can oxidize manganese to the non-available form. Culture assays and spectrophotometric analysis of crude culture extracts derived from Gaeumannomyces support the involvement of a multi-copper oxidase such as laccase, and possibly other extracellular oxidative enzymes, in manganese oxidation. Twelve putative laccase genes were found in Magnaporthe grisea, a fungus closely related to Gaeumannomyces graminis and we found strong expression of lcc 15 under manganese oxidizing conditions. Two genes with homology to manganese peroxidase also were identified in Magnaporthe grisea, and targeted deletion of these two genes resulted in greatly reduced virulence. It appears that there are at least two separate enzymes involved in manganese oxidation by these fungi that could influence their virulence and rhizosphere interactions via biological control. Efforts to further characterize metabolites produced by L. enzymogenes C3 that might be involved in biological yielded factors that have positive and negative effects on biocontrol efficacy. Strain C3 produces three ²1,3-glucanases and two chitinases, the production of which is regulated by a clp gene homologue belonging to the crp gene family. Using mutants deficient in either ²1,3-glucanase or chitinase activity or deficient in both groups of enzymes, ²1,3-glucanase was found to be involved in biocontrol of damping off of sugarbeet, caused by Pythium ultimum and leaf spot of tall fescue, caused by Bipolaris sorokiniana, while chitinase activity was shown to contribute to control of Bipolaris leaf spot. These enzymes, however, did not account entirely for biocontrol activity by C3. A small molecular weight, heat-stable antifungal factor, presumably involving a family of lipopeptide antibiotics, was found to cause abnormal hyphal growth in which hyphal polarity was disrupted. The factor was effective in vitro against all plant pathogenic fungi tested, as well as oomycetes. Using a mutant strain of C3 with reduced antibiotic excretion, evidence was found that the antifungal factor might have a role in biocontrol. Strain C3 induced systemic resistance in tall fescue against B. sorokiniana when the bacterium was applied to roots, while localized resistance was induced when C3 was applied to leaves. The same spatial phenomenon was found other strains of bacterial species were tested on tall fescue. Induced resistance by C3 can be elicited in wheat as well, being active against B. sorokiniana and Fusarium graminearum, but no evidence has been for induction of resistance in dicots by C3. Strain C3 produces a phytotoxic factor when grown in vitro or when applied to seed. The factor inhibits seed germination and reduces root growth in all graminaceous plants tested; some dicots (soybeans, tobacco) are sensitive to the factor, but sugarbeet is not. While the factor does not affect plants when it is applied to foliage, its effect on root growth is a concern when C3 is applied as a seed or root treatment.

Publications

Al-Naimi, F.A., K. A. Garrett, and W. W. Bockus. Competition, facilitation, and niche differentiation in foliar pathogens. Oecologia, In Press. Arriola, L., M.K. Hausbeck, J. Rogers, and G.R. Safir. 2000. The Effect of Trichoderma-harzianum and Arbuscular Mycorrhizae on Fusarium Root Rot in Asparagus. 2000. Hort. Tech. 10:141-144 Beattie, G. A. and L. M. Marcell. 2002. Comparative dynamics of adherent and non-adherent bacterial populations on maize leaves. Phytopathology 92:1015-1023. Blouin-Bankhead, S., Landa, B.B., Lutton, E., Weller, D.M., McSpadden Gardener, B. 2003. Minimal changes in rhizobacterial population structure following root colonization by wild type and transgenic biocontrol strains. FEMS Microbiol Ecol. submitted 12/18/03. Cox, C. M., K. A. Garrett, W. W. Bockus, and L. Fang. Reactions of tallgrass prairie grasses to tan spot and take-all pathogens from wheat and the potential for apparent competition. In Review Cox, C.M., K. A. Garrett, T. S. Cox, W. W. Bockus, and T. Peters. Reactions of perennial grain accessions to four major cereal pathogens of the Great Plains. In Review Cox, C. M., K. A. Garrett, and W. W. Bockus. Meeting the challenge of disease management in perennial grain systems. Renewable Agriculture and Food Systems: In Press. Cox, C.M., K. A. Garrett, R. L. Bowden, A. K. Fritz, S. P. Dendy, and W. F. Heer. 2004. Cultivar mixtures for the simultaneous management of multiple diseases: Tan spot and leaf rust of wheat. Phytopathology 94:961-969. Davelos, A. L., Xiao, K., Martin A. P., Samac, D. A., and Kinkel, L. L. 2004. Spatial variation in Streptomyces genetic composition and diversity in a prairie soil. Microb. Ecol. 39 (1): 1 - 11. Davelos, A. L., Kinkel, L. L., and Samac, D. A. 2004. Spatial variation in the frequency and intensity of antibiotic interactions among Streptomycetes from Prairie Soil. Appl. Environ. Microbiol. 70: 1051-1058. Davelos, A. L., Xiao, K., Flor, J. M., and Kinkel, L. L. 2004. Genetic and phenotypic traits of streptomycetes used to characterize antibiotic activities of field-collected microbes. Can. J. Microbiol. 50: 79-89. Del Rio, L.E., R. Henson, and T. Gulya. 2003. Use of Intercept for control of Sclerotinia on dry beans, canola, and sunflower in North Dakota. Proc. 2003 Sclerotinia Initiative Annual Meeting. Bloomington, MN 23pp. Del Rio, L. E., C. A. Martinson, and X. B. Yang. 2002. Biological control of Sclerotinia stem rot of soybean with Sporidesmium sclerotivorum. Plant Disease 86:999-1004. Gage, S.H., M. Colunga, G.R. Safir, J.J. Kelly, and A. Momin. 2001. A structural design for management and visualization of information from a crop simulation model applied to a regional scale. Computers and Electronics and Agriculture. 33:77-84 Gage, S., M. Colunga-Garcia, G. Safir, J. Qi, and A. Prasla. 2003. Development of a simulation environment for the management and visualization of information from simulation models applied to a regional scale. LTER Ecological Research , All Scientist Meeting. September 18-21, 2003, Seattle, WA. Poster presented. Gage, S.H., M. Colunga-Garcia, G.R. Safir. 2003. Application of climate and crop data into a framework for regional crop modeling. American Society of Agronomy-Crop Science Society of American-Soil Science Society of America meeting. November 2-6, 2003, Denver, CO. Garrett, K.A., S. P. Dendy, A. G. Power, G. K. Blaisdell, H. A. Alexander, and J. K. McCarron. 2004. Barley yellow dwarf disease in natural populations of dominant tallgrass prairie species in Kansas [Disease Note]. Plant Disease 88:574. Garrett, K.A., M. Kabbage, and W. W. Bockus. 2004. Managing for fine-scale differences in inoculum load: Seeding patterns to minimize loss to wheat take-all. Precision Agriculture 5:291-301. Garrett, K.A., L. V. Madden, G. Hughes, and W. Pfender. 2004. New applications of statistical tools in plant pathology. Phytopathology 94:999-1003. Garrett, K.A., M. Kabbage, and W. W. Bockus. 2003. Managing for fine-scale differences in inoculum load: seeding patterns to minimize loss to Gaeumannomyces graminis var. tritici. Precision Agriculture in press Grace, P.R., M. Colunga-Garcia, S. H. Gage, G.R. Safir, and G. P. Robertson. 2004. The potential impact of climate change on soil organic and agricultural management on carbon resources in the North Central Region of the United States. Ecosystems. (In Press) Gutierrez, L.J. and B.B. McSpadden Gardener. 2004. Identification and fungicide sensitivity of fungal pathogens causing tomato fruit rot in Ohio. Phytopathology 94:S37. Gutierrez, Laura J. 2004. Fungicide sensitivity and biological control potential of tomato fruit rot pathogens in Ohio. Master's Thesis. The Ohio State University, Department of Plant Pathology. Huber, D.M. 2003. Nutrient-Disease interactions in the take-all disease of cereals. Proc. Symposium on Take-all, April 2003, Temuco, Chili. Huber, D.M. 2003. The role of plant nutrition in disease. Proc. Symposium March 2003, Relacao entre Nutricao de Plantas e Incidencia de Doencas, Piracicaba, SP, Brazil. Jiang, W., K. A. Garrett, T. Harvey, and R. L. Bowden. The Window of risk for emigration of wheat streak mosaic virus varies with host eradication method. In Review. Kaitany, R., H. Melakberhan, G.Bird, and G. Safir. 2000. Association of Phytophthora sojae with Heterodera glycines nutrient stressed soybeans. Nematropica. 30:193-199. Kaitany, R.C., L.P. Hart, and G.R. Safir. 2001. Virulence composition of Phytophthora soja in Michigan. 2001. Plant Disease 85:1103-1106. Kilic-Ekici, O. and Yuen, G.Y. 2003. Induced resistance as a mechanism of biological control by Lysobacter enzymogenes strain C3. Phytopathology 93:1103-1110. Kilic-Ekici, O. and Yuen, G.Y. 2004. Comparison of strains of Lysobacter enzymogenes and PGPR for induction of resistance against Bipolaris sorokiniana in tall fescue. Biological Control 30:446-455. Kobayashi, D.Y., Reedy, R.M., Palumbo, J.D., and Yuen, G.Y. 2005. A clp gene homologue belonging to the Crp gene family globally regulates lytic enzyme production, antimicrobial activity, and biological control activity by Lysobacter enzymogenes strain C3. Appl. Environ. Microbiol. 71:261-269. Mathew, F.M. and L.E. del Rio. 2003. Development of a less complex medium for production of Sporidesmium sclerotivorum. On-line publication no. P-2004-0030-NCA. www.apsnet.org/meetings/div/nc03abs.asp Morgan, G. W., K. A. Garrett, T. C. Todd, and N. A. Tisserat. Effects of fungicides, burning and topographic position on dominant tallgrass prairie grasses and their pathogens. In Review American Midland Naturalist. McSpadden Gardener, B. 2003. Ecology of Bacillus species in agricultural soils. Phytopathology 93:S117. Abstract. McSpadden Gardener, B. 2003. Microbial biodiversity in agricultural systems: General and specific barriers to inoculation. Phytopathology 93:S100. Abstract. McSpadden Gardener, B. R. Joshi, L. Gutierrez, E. Lutton, and S. Grewal. 2003. Options and challenges for assessing microbial diversity in the phyllosphere. Phytopathology 92:S106. Abstract. Ortiz, O., K. A. Garrett, J. J. Heath, R. Orrego, and R. J. Nelson. 2004. Management of potato late blight in the Andean highlands: Evaluating the benefits of Farmer Participatory Research and Farmer Field Schools. Plant Disease 88:565-571. Palumbo, J.D., Sullivan, R. and Kobayashi, D.Y. 2003. Molecular characterization and expression in Escherichia coli of three ?-1,3-glucanase genes from Lysobacter enzymogenes strain N4-7. J. Bacteriol.: 185:4362-4370. Palumbo, J.D., Yuen, G.Y., Jochum, C.C, Tatum, K. and Kobayashi, D.Y. 2005. Mutagenesis of ²-1,3-glucanase genes in Lysobacter enzymogenes strain C3 results in reduced biological control activity towards Bipolaris leaf spot of tall fescue and Pythium damping off of sugarbeet. Phytopathology 95:(in press) Reedy, R.M. and Kobayashi, D.Y. 2003. Evidence for induction of the type III secretory pathway in the biocontrol bacterium Lysobacter enzymogenes strain C3 in the presence of fungal cell constituents. Phytopathology 93: S73. Rosenberg, M.S., K. A. Garrett, Z. Su, and R. L. Bowden. 2004. Meta-analysis in plant pathology for research synthesis. Phytopathology 94:1013-1017. Ryan, A. D., Kinkel, L. L., and Schottel, J. L. 2004. Effect of pathogen isolate, potato cultivar, and antagonist strain on potato scab severity and biological control. Biocontrol Sci. Techn. 14 (3): 301-311. Sabaratnam, S. and G. A. Beattie. 2003. Differences between Pseudomonas syringae pv. syringae B728a and Pantoea agglomerans BRT98 in epiphytic versus endophytic colonization of leaves. Applied and Environmental Microbiology 69:1220-1228. Safir, G.R., S.H. Gage, M.Colunga, and A. Momin. 2001. Biocontrol regional scales. 2001 Proc. Third Joint Reg. Mtg. (Biocontrol in a new millennium). Oct. 26-29, Estes Park., CO pp. 78-80. Safir, G. S. Gage, M. 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Impact Statements

Growers (particularly in NE, MN, KS, and IN) have new management strategies that are based on research done by the committee.
The experiments indicate that plant growth and yield can be improved using biocontrol bacteria as seed treatments. Further characterization of environmental variables ssociated with maximum yield responses will indicate which components of the soil and rhizosphere microflora should be managed for optimum crop productivity.
An increased understanding of the effects of biocontrol treatments at large scales (county-regional) over a period of years, accomplished through simulation of treatment effects on phenological parameters, such as leaf area and yield, of the crop in question should provide important information regarding the potential use of a biocontrol procedure at both biological and economic levels.
An understanding of the virulence mechanisms of soilborne plant pathogens is important for targeting with biological or cultural control practices. Offsetting virulence through the biological inhibition of essential extracellular enzymes, or by enhancing plant defense mechanisms, could reduce the 5 to 8% chronic losses caused by these pathogens.
Research conducted under this project will provide new information on the ecology, dynamics, and coevolution of plant-associated microbial communities in soil and on leaves in both agricultural and native habitats.
This research will also form the basis for developing integrated crop management systems that use non-pesticidal approaches for disease management. We have begun and examined the development of a framework for evaluation of farmer participatory research.