Brief Summary of Minutes of Annual Meeting

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.

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.

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.