Brief Summary of Minutes of Annual Meeting

Objective 1: To identify and characterize new biological agents, naturally suppressive soils, cultural practices, and organic amendments that provide control of diseases caused by soil borne plant pathogens. California (UCR - Borneman): Identifying Microorganisms Involved in Meloidogyne incognita Suppressiveness: This research will be accomplished through three Specific Objectives: (1) Identify Meloidogyne incognita suppressive soils through a survey of Californian soils; (2) Identify microorganisms whose populations positively correlate with M. incognita suppressiveness; (3) Validate the suppressive nature of the microorganisms identified in Objective 2 by introducing them into non-suppressive soils. Identifying M. incognita suppressive microorganisms will be an important discovery and a significant step towards the long-term goals of this project, which are to (a) understand how specific soils suppress M. incognita, (b) identify the parameters essential for successfully transferring M. incognita suppressiveness to non-suppressive soils, (c) utilize our fundamental understanding of these soils to develop novel strategies to manage M. incognita and, (d) create and implement predictive tools to guide cropping and pest management decisions. Identifying Microorganisms Involved in Replant Suppression: To accomplish this research, we are utilizing a population ecology strategy comprised of three phases: (I) utilization of OFRG to identify the microorganisms whose population levels correlate with the functional parameter through extensive rRNA gene analysis, (II) validation of the population trends with quantitative PCR and, (III) validation of the function of the organisms by reintroducing them into their natural environment. California (UCR - Stanghellini): 1. Insect control studies: Our objective was to determine if fungus gnats are involved in the acquisition and transmission of plant pathogenic oomycete propagules similar to that which has been documented for Fusarium avenaceum and Pythium aphanidermatum. Fungus gnat larvae were allowed to feed on oomycete propagules for 24 hours after which their intestinal tracts were microscopically examined for the presence of propagules. Chlamydospores of Phytophthora ramorum, hyphal swellings of Pythium splendens, P. sylvaticum and, P. ultimum were observed in the intestinal tracts as well as excreta of the larvae. Following excretion, propagules germinated within 24 hours. Additionally, chlamydospores of P. ramorum excreted by internally infested larvae were shown to infect and colonize detached rhododendron leaves. The role of fungus gnats and shore flies as vectors of the above-mentioned oomycete species are being studied. 2. N-Serve studies: Significant disease control (using cucumber plants as the host and Pythium aphanidermatum as the root pathogen in a model system) was achieved following amendment of the nutrient solution with various concentrations of N-Serve or Truban (marketed as Dwell, a nitrification inhibitor).The amendments, although exhibiting in vitro fungicidal activity, had no in vivo effect on the pathogens in the recirculating cultural system. Current data indicates that that these chemical amendments function by selectively stimulating the resident fluorescent pseudomonad population, which induce resistant mechanisms in the plant. There was a significant relationship between disease control and the population of fluorescent pseudomonads 3. Microbe-mediated germination of ascospores. Amending soil with diverse antibiotics which suppress Gram + bacteria, but not Gram  bacteria, suppressed ascospore germination in the rhizosphere of melon roots. These results support and extend previous studies with penicillin which implicated Gram + bacteria as a vital component in the induction of ascospore germination in the rhizosphere of melon root. Additionally, extracts from a soil which supports ascospore germination also restored germination of ascospores in sterilized soil. Nebraska (Univ. of Nebraska- Gary Yuen) Lysobacter enzymogenes strain C3, a bacterial antagonist of fungal pathogens, was found to be an antagonist of plant parasitic nematodes. Motile stages of plant-parasitic nematodes were inactivated and lysed when placed in cultures containing strain C3. Experiments with Heterodera schactii (sugarbeet cyst nematode) in growth pouches revealed root feeding and reproduction by the nematode was inhibited in the presence of C3, providing evidence that strain C3 may have potential as a nematode biocontrol agent. New York (Cornell- Christine Smart and George Abawi) A. Assessing and documenting the suppressive capacity of the soil under different production systems: In 1992 an 8-acre long-term cropping systems site was established at the Vegetable Research Farm at NYSAES, Geneva. When this research site was established, it was determined that the site was of uniform soil health before it was divided into four blocks representing four different production systems; conventional, organic, IPM present and IPM future. The conventional block was managed used standard management practices recommended in the Cornell production guidelines, in the IPM present block scouting and winter cover crops were used while in the IPM future block, season-long soil building crops were also incorporated into the vegetable rotation. The organic block was managed following certified organic production standards. From 2002 through 2006, soil samples collected from the four production systems were assessed for general soil suppressive capacity using the developed soil bioassay with bean. The soils managed using sustainable soil management practices in the future IPM and organically managed blocks (crop rotation, cover crops, and season-long soil building crops) were more suppressive to soil-borne pathogens affecting vegetables including Rhizoctonia, Pythium, Thielaviopsis, and Fusarium than the conventional block. Yield differences were also observed. In 2006 four, eight-row sections of beans were planted in each production systems block. At harvest, total weight was determined for two 10 ft sections of bean plants per plot per production system then the pods were removed and weighted from a 5lb sub-sample per plot. The IPM systems had the highest total plant weight than the conventional and the organic systems. Ohio (Ohio State- Brian McSpadden-Gardener) The influences of different organic transition strategies on soil chemistry and soilborne disease suppressiveness were studied. The strategies included tilled fallowing, and the production of mixed hay, open-field vegetables, and high tunnel vegetables. Replicate soil samples were removed twice from the field site in 2005 and assayed in the growth chambers. Significant increases in soil nutrients and pH were observed in all of the compost-amended soils; however, no significant changes in soil chemistry were observed among the treatments in the unamended soils. The effects of the different treatments on plant health were assayed on tomatoes and soybeans, in the presence and absence of pathogen inoculum (Pythium ultimum and/or Phytophthora sojae). Overall, compost amendments led to decreased plant stand but increased vigor of the surviving seedlings in both crops, indicating that increased disease pressure was offset by increased soil fertility. When comparing transition strategies, suppressiveness to damping-off in tomato was greatest in the hay treatments, regardless of compost amendments or pathogen pressure. Results were similar for soybeans grown in the presence of inoculum. These results indicate that transition management strategies can significantly impact soil quality and biologically-based soilborne disease suppressiveness in subsequently planted crops. Microbial populations were analyzed in the rhizosphere of tomato (cv. Tiny Tim) and edamame soybean (cv. Sayasume). For tomato, significantly higher root colonization rates of Pseudomonas spp. were observed with compost amendments under all cropping strategies. In addition, management strategy effects were determined, with lower colonization levels in the tilled fallow. On edamame soybeans, similar trends were observed in relation to total cultured Pseudomonas populations. For the hay treatment, compost addition resulted in more abundant rhizosphere populations of 2,4-diacetylphloroglucinol-producing pseudomonads of both crops. Bacterial community profiling using terminal restriction fragment length polymorphism (T-RFLP) revealed ribotypes specifically associated with each cropping system. Characterizing selected ribotypes could lead to the identification of novel microbial populations associated with disease suppressiveness in these fields. Oregon (Oregon State- Parke and Grunwald) Root infection by Phytophthora ramorum: We are investigating the soil ecology and epidemiology of Phytophthora ramorum, cause of sudden oak death and ramorum blight. This was previously believed to be a pathogen capable of infecting only above-ground plant parts. We demonstrated that potted rhododendron plants can become infected through roots if plants were grown in artificially infested potting media. This year, we investigated the possibility that forest trees could become infected through their root systems. Roots of young tanoak (Lithocarpus densiflorus) trees growing in a naturally-infested forest in Humboldt Co., California were found to be infected with P. ramorum. This finding could drastically alter disease management efforts as it suggests that movement of infested soil could spread the disease to new locations. Epidemiology and control of Phytophthora species in recirculating irrigation water: We are continuing our studies on the detection, epidemiology, and control of Phytophthora species in recirculating irrigation systems. We conducted dose-response studies with P. citricola on rhododendron as a prelude to testing the efficacy of water treatment methods for the elimination of Phytophthora species in irrigation water. Systems approach for producing nursery stock free of plant pathogenic Phytophthora species: In a multi-year project started this year, we are testing a new approach to ensure that nursery stock is free from quarantine pests and pathogens. This systems approach is based on the Hazard Analysis Critical Control Points (HACCP) strategy for identifying and mitigating sources of contamination, similar to the approach for eliminating contaminants during food processing. Initially our study is designed to focus on the model pathogen Phytophthora but the project may be expanded in the future to include other pathogens and pests. In the first year of the study, we designed and implemented a sampling strategy to detect sources of Phytophthora contamination in four nurseries and are identifying the critical control points for each. We have processed 737 plant, soil, and water samples from all stages of nursery production, and approximately 150 putative Phytophthora isolates are being identified to species by direct sequencing of the internal transcribed spacer regions (ITS-1 and ITS-2) in the Grunwald lab (USDA-ARS, Corvallis, OR). In the second year, we will recommend specific changes in nursery management practices to correct the problems. In the third year, we will sample again to determine if sources of contamination have been eliminated. Washington (USDA-ARS, Pullman, Paulitz, Okubara, and Weller) 1. Pseudomonas spp. that produce phloroglucinol and are responsible for take-all suppression in wheat, do not suppress Pythium root rot. To assess the role of DAPG producers in the suppression of take-all, soils that had undergone wheat or pea monoculture, and a non-cropped soil were infested with the take-all pathogen. A greater suppression of take-all was observed in wheat seedlings grown in soils from continuous wheat or pea monoculture fields than in soil from non-cropped fields. Pasteurization of the pea monoculture soil resulted in a loss of DAPG producers and concurrent inability to suppress take-all. Strains of DAPG-producing P. fluorescens inhibited in vitro mycelial growth of Pythium spp.; however, TAD soils from Lind and Quincy, harboring large populations of indigenous DAPG-producing P. fluorescens, were not suppressive to Pythium root rot. Pythium root rot severity was similar on Penawawa wheat seedlings grown in soils conducive or suppressive to take-all infested with Pythium spp. Pythium spp. were less sensitive to synthesized DAPG than the take-all pathogen, which may explain the lack of Pythium suppression. In greenhouse experiments, wheat seeds treated with different DAPG-producing P. fluorescens genotypes or the recombinant strains Z30-97 or Z34-97, able to produce phenazine-1- carboxylic acid and DAPG, did not reduce Pythium root rot on wheat seedlings. 2. Fallow from mechanical tillage or herbicide results in transient reduction in Rhizoctonia activity. There is no resistance to Rhizoctonia root rot of cereals or chemical controls. Rhizoctonia survives as mycelium in roots of volunteer cereals and grassy weeds. Growers in dry areas fallow the soil to conserve moisture for the following year, but fallow with mechanical tillage leads to wind erosion of soil. We investigated more environmentally- sound methods of fallow using herbicides or reduced mechanical fallow (with inverted sweeps), to see if the activity of Rhizoctonia was reduced. Activity was highest in March-May, when soil moisture was optimal in the upper soil layers; and declined until Sept. There was no effect of fallow on R. solani until the end of the growing season, when activity was lowest in the mechanical fallow treatment. However, activity of R. oryzae was lower in the fallow treatment throughout the growing season. In the following season, differences between continuous cropping and fallow diminished. Rhizoctonia solani and R. oryzae can survive in the absence of a crop, although their activity is diminished. 3. Composition of Pythium spp. in cereal cropping systems is influenced by crop rotations and fallow We conducted a survey from 41 sites in Eastern Washington in 2005, and quantified up to 8 Pythium species from a single soil sample. Conventional dogma says that cropping systems should not affect Pythium populations, because of the wide host range of most Pythium species. However, we found that P. ultimum and P. irregulare were mostly associated with a wheat-lentil rotation, and although the most pathogenic, were the least frequently isolated. P. irregulare group IV and P. abappressorium were the most frequently isolated, and were also in the highest concentration. The abundance of Pythium spp. in a given site showed a bimodal distribution- locations in higher rainfall zones had 6-9 species, while only 0-2 species per site were isolated from drier locations. In general, fallow reduced the population of Pythium spp. to below the detection limit (10 cfu/g, or about 10 fg DNA/sample), but P. abappressorium and P. irregulare group IV were not eliminated by fallow. In conclusion, composition of Pythium spp. and populations in soil are influence by cropping practices in cereal production. Objective 2 Understand how microbial populations and their gene expression are regulated by the biological (plants and microbes) and physical environment and how they influence disease. Arizona (Univ. of Arizona- Pierson): Characterization of the molecular mechanisms responsible for phenazine gene regulation in P. aureofaciens 30-84 is continuing to provide insights into how a root-associated bacterium senses its environment and responds by altering patterns of gene expression. We identified multiple regulatory networks that govern phenazine production. These systems include the GacS/GacA two component system, the RpoS stationary/starvation sigma factor, the PhzR/PhzI quorum sensing (QS) system, the RpeA/RpeB negative two component regulatory system and an rsmZ/RsmA post-transcriptional control mechanism. We also study the role of cross-communication among rhizosphere bacteria on phenazine gene expression in strain 30-84 and the effects of the indigenous wheat rhizosphere community on this expression. 1. Molecular analysis of negative signaling among wheat rhizobacteria. We have focused on the wheat rhizosphere colonizer strain PU-43. Strain PU-43 produces at least two diffusible signals, one is an AHL that can complement a phzI mutant of strain 30-84 while the second more polar compound blocks phenazine gene expression in strain 30-84 and b-galactosidase activity in the genomic reporter strain 30-84Z. Currently we have extracted the negative signal into butanol and are attempting further structural characterization. We also developed a medium throughput colorimetric assay for negative signal production by PU-43 and have constructed ca. 6,000 transposon insertions in the strain. Using this assay, we have ca. 50 insertions in strain PU-43 that result in the loss of negative signal production. Several of these inserts have been cloned and sequenced and we are currently obtaining the wild type versions of these genes for complementation analysis. 2. Biofilm development requires the PhzR/PhzI, CsaR/CsaI and PhzB. Our studies on the role of QS in strain 30-84 resulted in the discovery that the PhzR/PhzI QS system is important in biofilm initiation. Of greater excitement is the fact that a phzB mutant of strain 30-84 that is specifically blocked solely in phenazine production also fails to form biofilms. Complementation of the phzB mutant with extra copies of phzR/phzI has no effect but complementation with phzB fully restores biofilm development. Introduction of plasmid pLSP10-30F in which the phzFABCDO genes are expressed from the E. coli lac promoter resulted in earlier and more dense biofilm development in the phzB mutant than the wild type strain. 3. Characterization of the RpeA/RpeB negative two component regulatory system in strains 30-84 and P. aeruginosa PAO1. RpeA/RpeB is a sensor kinase/response regulator system that couples aspects of cell metabolism to secondary metabolite production. Mutations in rpeA or rpeB result in higher levels of phenazine gene expression in minimal medium in comparison to the wild type strain. Interestingly, a phzR/rpeA double mutant defective in both the negative two component system and QS produces phenazines at the same level as the wild type in all media. Thus, the loss of rpeA bypasses the need for QS in strain 30-84. We have shown that mutations in P. aeruginosa PAO1 rpeA and rpeB homologs also resulted in higher than wild type levels of pyocyanin production, suggesting this regulatory system may be universal among phenazine producers. Nebraska (Univ. of Nebraska- Gary Yuen) Lysobacter enzymogenes strain C3 produces an antibiotic that affects sphingolipid production in fungi. The antibiotic was found to be dihydromaltophilin, which was first reported in a Streptomyces sp. and is structurally related to xanthobaccin A produced by a different Lysobacter strain. The gene cluster responsible for biosynthesis of the antibiotic in C3 was identified. The cluster includes a gene encoding a hybrid polyketide synthase-nonribosomal peptide synthetase. Disruptions of either portion of the gene resulted in loss of production of the antibiotic by C3. The mutation also resulted in significantly reduced biocontrol activity against Bipolaris sorokiniana on tall fescue as compared to wildtype C3, providing evidence that the antibiotic has a role in biocontrol. The role of the antibiotic in control of Fusarium graminearum in wheat by C3, however, is uncertain as the mutant strains disrupted in antibiotic were inconsistent in their effects on Fusarium head blight in repeated experiments. New York (Cornell- Christine Smart and George Abawi) A. Evaluation of a biological nematicide for use as a seed treatment against northern root-knot nematode on vegetables: We have previously shown that STAN (a seed treatment with activity against nematodes) is effective against both the root-knot and lesion nematodes. This biological nematicide has been incorporated as a component along with an insecticide and fungicide as a combination seed treatment called AVICTA. The AVICTA seed treatment was highly effective against Meloidogyne hapla as evidenced by the extremely low number of juveniles of M. hapla observed in tomato roots and resultant root-galls at three weeks after planting. These treatments appeared more effective even than the standard soil treatment with Vydate (oxamyl). The lower efficacy of Vydate observed in this trial might be due to the frequent watering of the small containers used thus potentially leaching out the nematicide. However, growing tomato seeds treated with AVICTA in plugs for four weeks before transplanting into field microplots infested with M. hapla did not protect the plants from infection and damage by the nematode. It is not known if the nematicide seed treatment was leached out or remained in the plug in an active or inactive form. Vydate soil application significantly reduced root-galling severity ratings and reproduction of M. hapla on roots and in soil, but it did not improve yield in comparison to the yield of tomato grown in the methyl-bromide treated plots. B. Utility of plant growth-promoting rhizobacteria for disease control of foliar pathogens: Plant activators provide promising disease management options by activating plant defense responses via the systemic acquired resistance (SAR) and/or induced systemic resistance (ISR) pathways. While the SAR pathway has been extensively studied in the greenhouse, less is known about ISR. The goal of this research was to determine how SAR and ISR individually or in combination alter tomato host responses to the foliar bacterial pathogen Pseudomonas syringae pv. tomato. Marker genes for the salicylic acid, jasmonic acid, and ethylene pathways were followed using real-time quantitative PCR. Results revealed that application of ISR-inducing Bacillus spp. prior to induction of SAR did not delay the onset of SAR nor did it modify disease control. Plants treated with ISR + SAR inducers or SAR inducers alone had a significant increase in expression of the acidic PR-1 gene and also had a high level of disease control. Pathogen inoculation induced the jasmonic acid pathway, though expression of the marker gene PinII was significantly lower in SAR-induced plants. The ISR-inducing Bacillus spp. ineffectively managed disease. Ohio (Ohio State- Brian McSpadden-Gardener) Different farm management practices that significantly affect crop health also alter the abundance of specific populations of bacteria that can suppress plant pathogens. Pseudomonas spp. that produce the antibiotic 2,4-diacetylphloroglucinol (DAPG) represent one such population of biocontrol bacteria, and the phlD gene, which is essential for the production of DAPG, can be used to monitor their abundance. The incidence and relative abundance of root-colonizing phlD+ Pseudomonas spp. were influenced by crop rotation, tillage, organic amendments, and chemical seed treatments in subtle but reproducible ways. In no-till corn plots, two-year rotations with soybeans resulted in plants with two-fold fewer phlD+ pseudomonads per gram of root, but three-year rotations with oats and hay led to population increases of the same magnitude. Interestingly, tillage inverted these relationships in two consecutive growing seasons, indicating a complex but reproducible interaction between rotation and tillage on the abundance of DAPG-producers. Similar changes in relative abundance were noted following very heavy applications (12 T per ha) of composted dairy manure, but not at a more typical application rate (6 T per ha) despite substantial changes in soil nutrient status and plant vigor that occurred at the lower rate. Chemical seed treatments intended to control fungal pathogens and insect pests also led to significantly more abundant populations of phlD regardless of soil type. Within a treatment, the correlation between the abundance of phlD+ Pseudomonas spp. and the crop stand and yield was generally positive in corn, and the strength of those correlations were greater in continuous corn plots where there was more root disease. In contrast, such correlations were generally negative in soybeans, a difference that may be partially explained by difference in application of N fertilizers and soil pH. In total these data indicate that farm management practices that alter soilborne root disease pressure also alter the abundance of biocontrol bacteria that can suppress their activities in a predictable, context-dependent manner. Oregon (USDA-ARS, Corvallis, Loper) A new class of cyclic lipopeptides was discovered by purifying the natural product associated with a secondary metabolism region of the genome of Pseudomonas fluorescens Pf-5. The orphamides, founding members of the new class of lipopeptides, were purified, their structures were determined, and their activities against an Oomycete plant pathogen were established. The genomisotopic approach, which employs a combination of genomic sequence analysis and isotope guided fractionation to identify unknown compounds, represents an efficient new method that will allow scientists to tap the tremendous source of novel and possibly bioactive natural products that can be discovered from genomic sequence data. With a group of natural products chemists from the Scripps Institution of Oceanography in San Diego, California, we applied the genomisotopic approach for the first time, which resulted in the discovery of orfamide A. Orfamide A production by strain Pf-5 was shown to play a key role in motility of the bacterium and lysis of zoospores of a phytopathogenic Phytophthora sp. The results of this study include the discovery of a new class of compounds that suppress an important group of plant pathogens and could contribute to biological control of plant disease, and a new approach that will advance the discovery of new natural products from the rapidly expanding numbers of organisms whose genomes are being sequenced. Washington (USDA-ARS, Pullman, Paulitz, Okubara, and Weller) 1. Development of real-time PCR for quantification of DAPG genotypes of Pseudomonas fluorescens. Previous studies on the ecology of DAPG (2-diacetylphoroglucinol)-producing Pseudomonas spp. have relied on a combination of culturing and PCR identification. In the late 90s, our unit developed a PCR-based terminal-dilution endpoint assay. Bacteria are washed from roots, and dilutions are made in microtiter dishes containing a nutrient medium. Specific genotypes in the endpoint dilution can be identified by PCR and RFLP. But this method cannot quantify a mixture of genotypes, since only the dominant one in the end dilution will be detected. Genotype-specific primers were developed from sequences of the phlD gene for the A, B, D and I genotypes. PCR efficiencies ranged from 80 to 98%, and detection limits ranged from 5 to 298 cfu/reaction. However, detection limits were log 4.2 cfu/g of root, higher than the log 3.2 limit with the terminal dilution method. PCR efficiencies need to be further optimized. The real time PCR method has several advantages over the present method- it is culture independent; allows detection of dominant and subdominant genotypes; allows detection of indigenous bacteria (i.e. those not tagged with antibiotic resistance); and has a turnaround time of only 2 days, as opposed to 5 days with the other method. The quantitative PCR method is less labor intensive, with slightly higher material costs per reaction. 2. Effect of Pseudomonas fluorescens isolate Q8r1-96 on gene expression in wheat roots A series of experiments was conducted with microarrays. Wheat cultivars Finley and 442 and 443 (near-isogenic lines that differ in the vrn1A vernalization/cold tolerance locus) were colonized with Pseudomonas. A number of genes were significantly up-regulated in the root, including genes for oxidative stress, ABA signaling, apoptosis, JA-mediated defense (PR-10a), and JA signaling (COI 1). Genes were up-regulated by 2.2 to 3.9 fold, and many were confirmed with real-time PCR Objective 3 To develop and implement biological control in agriculture. New York (Cornell- Christine Smart and George Abawi) For a biological control agent to be utilized in the field, it must be compatible with other products commonly used by growers. To determine if induced systemic resistance (ISR)-inducing biocontrol agents are compatible with other systemic acquired resistance (SAR)-inducing compounds, we are studying the effect of multiple plant activators on plants in the field. It is known that there is cross-talk between plant defense responses, and it is important to determine if there are interactions between products in the field. Utilizing both SAR and ISR-inducing compounds, we have found that SAR-inducing compounds are more effective at foliar control of bacterial speck on tomato (caused by Pseudomonas syringae pv. tomato). Additionally, we have found that in some years, ISR-inducing Bacillus sp. have increased yield in fresh market tomato varieties. Importantly, when both SAR and ISR compounds are applied to the tomato plant, there is no reduction in disease control. That is, the induction of ISR does not reduce the ability of SAR to control foliar diseases. We are currently isolating RNA from field samples to determine gene expression patterns for marker genes of three plant defense response pathways. This information will enable us to determine how, at the molecular level, plants are responding to multiple plant activators. Ohio (Ohio State- Brian McSpadden-Gardener) In Ohio, evaluations of bacterial seed treatments were performed on six conventional and four organic farms in 2005. The bacteria used were all Pseudomonas spp. that produce 2,4-diacetylphloroglucinol, a trait marked by the phlD gene. Soilborne pathogens were considered to be omnipresent, as isolates of pathogenic oomycetes (e.g Pythium spp. and Phytophthora sojae) and/or fungi (e.g. Fusarium spp. and Rhizoctonia solani) have been recovered from every field sampled by our laboratory. On conventional farms, average yields were higher for treated plots as compared to untreated plots at five of six locations, and, overall, average yields of biologically- and chemically-treated seed were higher than those of untreated seed by 1.2 and 1.4 bu / A, respectively, but such differences also were not statistically significant (P > 0.20) at the level of replication examined. On organic farms, biologically treated seed yielded 1.5, 4.6, 0.5, and 3.7 bu/A more at sites 1,2,3, and 4, respectively, but such differences were not statistically significant at the individual site level (P > 0.3 for each location) due to limited replication in the field designs. When considering the entire data set though, the bacterial seed treatments increased the average soybean yield (P < 0.10) across the state on certified organic farms. Across these four sites, yields were increased by 8% on average. V. Collaborations with other W-1147 members James Borneman (CA) is collaborating with Michael Stanghellini to identify microorganisms involved in the germination of Monosporascus cannonballus ascospores; with Ole Becker to identify microorganisms involved in (i) Meloidogyne suppressiveness and (ii) replant suppression of walnut and peach.; and with Don Cooksey to identify microorganisms involved in suppressing Pierces disease. L. S. Pierson (AZ) is collaborating with D. Weller and L. Thomashow at Washington State University and with J. Loper at Oregon State University on specific projects and with each member of the W-1147 team on general projects. Gary Yuen (NE) - Soil, plant and water samples will be provided by other W1147 members for the isolation of Lysobacter strains; and Lysobacter enzymogenes C3 will be provided to W1147 members for evaluation of biocontrol efficacy on different pathosystems. Brian McSpadden-Gardener (OH) has worked with Dr. Borneman on further development of a community structure-based analyses and approaches to identifying novel biocontrol agents. Jennifer Parke (OR) will investigate the possibility of collaborating with Mike Stanghellini on water treatment methods to control Phytophthora spp. in recirculating irrigation systems and will continue collaboration with N. Grunwald on the clean nursery stock project. Paulitz (WA) has initiated a collaboration with J. Borneman on how cultural methods affect microbial populations in no-till cereal production, and how a microflora suppressive to Rhizoctonia may develop during long-term no-till. The unit at Pullman, WA has a long collaboration with S. Pierson at Univ. of Arizona in studying phenazine biosynthetic pathways, and with J. Loper at USDA in Corvallis, on antifungal compounds produced by Pseudomonas spp.