Annual/Termination Reports:

[11/27/2004] [12/20/2004] [01/19/2006] [12/31/2006] [01/09/2008]

Report Information

Participants

J.H. McBeath, P. Gay - University of Alaska, Fairbanks;

L.S. Pierson III - The University of Arizona;

K. Subbarao - University of California, Davis;

J.O. Becker, J. Borneman,
J.A. Menge,
M. Stanghellini - University of California, Riverside;


C. Bull,
F. Martin - USDA-ARS Salinas;

W. Chun, University of Idaho;

N.W. Callan - WARC, Montana State University;

N. Goldberg - New Mexico State University;

G.S. Abawi - NYSAES, Cornell University;

J.E. Loper - USDA-ARS;

J. Parke,
F. Crowe - Oregon State University;

D.M. Weller,
T. Paulitz,
P. Okubara,
N. Grunwald - USDA-ARS;

E. Riga - Washington State University

Brief Summary of Minutes

1) Meeting brought to order by Chairman Sandy Pierson at 8:30 AM.

Present: J.H. McBeath, P. Gay, L.S. Pierson III, J.O. Becker, J. Borneman, J.A. Menge,
M. Stanghellini, N.W. Callan, G.S. Abawi, D.M. Weller, T. Paulitz. Absent: K. Subbarao, C. Bull, F. Martin, W. Chun, N. Goldberg, J.E. Loper, J. Parke, F. Crowe, P. Okubara, N. Grunwald, E. Riga.

2) Minutes from the previous technical meeting were discussed and approved.

3) W-1147 renewal. Administrative Advisor Don Cooksey reported that W-1147 had been approved for five years. The next renewal will be in 2007.

4) W-1147 Website. A website could be used to distribute state research reports and post a list of tested biocontrol agents. Several options for a W-1147 website were discussed. Don Cooksey will talk to Wesley Chun, and Sandy Pierson will talk to Ole Becker.

5) Meeting adjourned at 10:30 AM for a tour of MSU Plant Growth Center with Don Mathre.

6) Meeting resumed after lunch.

7) Membership. There was a discussion of increasing attendance and recruiting new members. It was suggested that we try to coordinate the annual meeting date with another scientific meeting.

8) Reports. All members must submit reports, whether attending the meeting or not. Don Cooksey instructed the committee to use the template that had been distributed and is available at http://www.escop.msstate.edu/archive/SAES422preparation.htm. The current Chair (Sandy Pierson) will incorporate all individual reports into the new annual report format.

9) Biocontrol Agent List. Members should send name of organism, pathogens controlled, usual host plant, and descriptive information to Sandy Pierson for eventual posting on a W-1147 website.

10) Meeting Locations. The 2004 meeting is to be a joint meeting with S-302 and NC-125. James Borneman will contact the other committees to determine the meeting arrangements. The 2005 meeting will be held in Portland, Oregon.

11) New Officers.

2004: Chairman, Nancy Callan; Secretary, James Borneman

2005: Chairman, James Borneman; Secretary, Tim Paulitz

12) Meeting adjourned at 6:00 PM, 25 October, 2003. Discussions continued over dinner.

Submitted by Nancy W. Callan, Secretary

Accomplishments

The complete annual report of accomplishments from W1147 for 2003 can be obtained from the Administrative Advisor. An abbreviated report follows.<br /> <br /> Goal 1: To identify and characterize new biological control agents, naturally suppressive soils, cultural practices, and organic amendments that provide control of diseases caused by soil borne plant pathogens.<br /> <br /> One of the challenges to improving biological control of soil borne plant diseases is to overcome the inconsistencies associated with host and pathogen complexity and diversity, the effects of competing microflora, and variations in soils and cultivation practices. The greatest strength of the regional research approach by members of W-1147 is the diverse expertise each member contributes. Since plant-pathogen systems are so varied, they must be tailored to each geographical area, crop variety, and pathogen. However, our collaborative approach can reveal similarities and common themes not otherwise noticeable. Members this year are working on 7 different plant pathogens, including bacterial, fungal and nematode pathogens. Current exciting results included the discovery of 23 potential biological control agents. A summary of this past years discoveries is given below.<br /> <br /> Intended outcome: The identification of new potential biological control agents.<br /> <br /> Milestone: Development of a new method for identifying suppressive organisms.<br /> <br /> Several W-1147 members are working to: (i) develop new experimental approaches for identifying microorganisms involved in pest suppressiveness in soil and, (ii) use this approach to identify bacteria and fungi involved in soil suppressiveness against several plant pathogens, including the plant-parasitic nematode Heterodera schachtii,and the fungus Phytophthora cinnamomi, the causal agent of avocado root rot. The hypothesis being tested is that the microorganisms responsible for disease suppression can be identified by correlating their population levels using molecular techniques with soil suppressiveness. This approach has four phases. The first phase is to create a series of samples exhibiting various levels of suppressiveness through manipulation of microbial community composition. In phase two, we utilize our newly developed, array-based method to identify microorganisms that correlate with suppressiveness. Phase three employs quantitative PCR to confirm the correlations between the microbial population trends and suppressiveness. In phase four, the candidate organisms are isolated and reintroduced to assess their abilities to produce suppressiveness.<br /> <br /> Results obtained this year demonstrated that this approach was successful in identifying organisms involved in H. schachtii suppressiveness.<br /> <br /> Milestone: Development of a real time fluorescent PCR assay specific for Aphanomyces euteiches. A. euteiches causes severe root rot of peas. Resistance is limited in commercial pea cultivars. Lack of progress in breeding for resistance may in part be due to limited discriminatory power of typical screening procedures, which rate disease severity using an integer scale. We developed a real time fluorescent PCR assay specific for A. euteiches.<br /> <br /> Milestone: Develop methods to estimate genetic diversity of pathogen populations in the field. When analyzing plant pathogen and microbial populations in the field, it is important to be able to estimate the level of genotypic diversity. The degree of variation for genotype and pathogenicity in a population of Aphanomyces euteiches within two growers fields with a history of Aphanomyces root rot was determined. We evaluated two hypotheses: (1) populations of A. euteiches are diverse genotypically and phenotypically within single fields, and (2) that populations of A. euteiches from different fields are well differentiated.<br /> <br /> Milestone: To measure the distribution and stability of naturally suppressive soils. A survey of California avocado groves is continuing to identify local groves, which have soils that are suppressive to Phytophthora cinnamomi. We are utilizing several experimental approaches to extract biological control agents from local suppressive soils. One of them is to identify bacteria and fungi from soil mixtures with varying degrees of suppressiveness. Molecular examinations of the microbial communities confirmed Galactomyces as a major component of the suppressive soil.<br /> <br /> A second approach is to characterize microorganisms spatially and temporally present in various stages of a field epidemic. Populations of P. cinnamomi appear to decline precipitously immediately behind the leading edge of the epidemic in some groves. Biocontrol fungi are being isolated from this region. We postulate that microorganisms, which fill a niche similar to that of the pathogen or that compete for chemical substrates important to the pathogen, such as root exudates, will be effective biological control agents. To identify such organisms, we have developed an in situ, culture-independent strategy to identify bacteria and fungi that rapidly grow in response to specified chemical substrates in environmental samples.<br /> <br /> Milestone:An additional approach to discover new biological control organisms is to identify suppressive organisms from sites where the pathogen has been around the longest. The rain forest of Papua New Guinea is thought to be the center of origin for P. cinnamomi. We have enlisted the aid of Bob Tombe, University of Goroka, Papua New Guinea, to cooperate with us and send samples every two months for the next two years. Unfortunately Dr. Tombe was robbed and lost his rented vehicle while collecting samples for us, so the cost and effort to get these samples has been great. We received only two new shipments of soil from New Guinea. Results from these shipments were rather disappointing with only a few soils showing suppressiveness to Phytophthora cinnamomi. These results were far less impressive than results we obtained last year. However, a group of these soils from the Mt. Kubor region of New Guinea gave exceptional suppressiveness. Phytophthora placed in these soils became vacuolated and died very quickly. Even chlamydospores were killed.<br /> <br /> Milestone: Determine the efficacy of biological control agents in the field. The efficacy of biocontrol agents was tested on both Sclerotinia sclerotiorum and S. minor at the Desert Research & Extension Center in El Centro, CA. Treatments evaluated were Plant Shield (Trichoderma harzianum), Companion (Bacillus subtilis), Contans (Coniothyrium minitans), and Rovral. None of the treatments including Rovral was effective against S. minor. These treatments were also duplicated in Yuma, AZ, and even at this site, all treatments failed. Incidence of lettuce drop caused by this species in different plots at El Centro varied between 60-80%. Similarly, of all the treatments against S. sclerotiorum, Contans provided almost complete control of lettuce drop. Less than 5% of the plants in plots treated with Contans were diseased. Studies are underway to compare the efficacy of Abamectin, a secondary product of Streptomyces avermitilis as a seed treatment against the root-knot and lesion nematodes Meloidogyne hapla and Pratylenchus penetrans, respectively.<br /> <br /> Milestone: The purpose of this work was to compare Rhizoctonia populations at different positions within the patch and at different soil depths and to see if patches would be maintained in the R. solani-infested cores over successive plantings in the greenhouse. At the first planting, activity of R. solani was higher in the center and inside edge, but after the second planting, there were no differences among the patch positions. Based on plant height, patches were maintained in only 6 out of 16 sets of cores. R. solani activity was similar at all soil depths from 2-20 cm.<br /> <br /> Evidence for suppression of Rhizoctonia in spring wheat after spring barley has also been documented in a 5 year rotation study at Ritzville. The area in patches in spring wheat following spring barley is less than half, compared to continuous spring wheat Maps also show that patches disappear and new ones appear on an annual basis. We will do further studies with this mapping data to see how many of the patches decline in a given year in each rotation.<br /> <br /> Milestone: Include analysis of the plant host for improving disease control. Potatoes (Solanum tuberosum) contain products termed osmotins, a family of proteins correlated with the plants survival against biotic and abiotic stresses. Osmotins have been implicated in broad-spectrum pathogen resistance as well as drought and cold tolerance. Although osmotin genes have been characterized from wild Solanum species, very little is known about osmotin genes from cultivated Solanum tuberosum. Sequence alignment revealed a range of 91-99% similarity to S. commersonii osmotin genes. A phylogenetic tree was generated for cultivar comparison based on osmotin gene amplified products.<br /> <br /> Milestone: Determine the effect of Brassica residue on the incidence of soil-borne diseases in chile. The primary objectives of this project were to compare disease incidence and overall yield of Phytophthora root rot, Verticillium wilt and Root-knot nematode in field plots treated with various Brassica crop residues.<br /> <br /> Goal 2: To understand how microbial populations and their gene expression are regulated by the biological (plants and microbes) and physical environment and how they influence disease.<br /> <br /> Intended outcome: Further understanding of the molecular basis of biological control mechanisms and the influence of the environment on the expression of these mechanisms.<br /> <br /> Milestone: Continue to elucidate the molecular basis of secondary metabolite production responsible for pathogen inhibition by Pseudomonas aureofaciens 30-84. <br /> <br /> Milestone: Delineate the phenazine biosynthetic pathway. <br /> <br /> Milestone: Characterize the molecular mechanisms by which T. atroviride controls plant pathogens. <br /> <br /> Milestone: Characterize the role of DAPG (Phloroglucinol) in control of Pythium spp. <br /> <br /> Milestone: Develop a technique to accurately quantify Pythium damage (root scanning with WinRhizo software). <br /> <br /> Milestone: Determine the effect of the addition of wild type and transgenic biocontrol strains on the indigenous rhizobacterial population. <br /> <br /> Milestone: Determine the host contribution to the process of root colonization. <br /> <br /> Milestone: Determine additional genes that contribute to rhizosphere success. <br /> <br /> Milestone: For the first time ever, the full genomic sequence of a biocontrol agent, Pseudomonas fluorescens Pf-5, was determined, in a collaborative project involving several W147 members (Loper, Pierson, and Thomashow). <br /> <br /> Milestone: Factors influencing the production of pyoluteorin by the biological control agent Pf-5 include autoinduction. <br /> <br /> Goal 3: To develop and implement biological control in agriculture.<br /> <br /> Intended outcome: To move basic research results into sustainable agriculture.<br /> <br /> Milestone: Utilize the bacterium Pseudomonas putida to control Phytophthora cinnamomi in the field. <br /> <br /> Milestone: The EcoSoils Bioject machine has been thoroughly tested and has been found to produce high quality biocontrol agent inoculum in the field and deliver it reliably in the irrigation water. <br /> <br /> Milestone: Determine the mechanism by which the cover crop Sudan grass reduces pathogen success.<br /> <br /> Milestone: Develop assessment methods for soil health. <br /> <br /> Milestone: Develop disease management strategy for the control of vine-decline of melons caused by the root-infecting fungus Monosporascus cannonballus.<br /> <br /> Milestone: Determine whether LCF (Liquid Compost Factor) enhanced chickpea germination in the field. <br /> <br /> Milestone: Pseudomonas corrugata is basically a weak pathogen of very little interest to plant pathologists. However, it has great potential as a biological control agent for fungal root diseases. <br /> <br /> Milestone: Reduction of stolon decay in Peppermint.

Publications

Impact Statements

1. Development of a new method for identifying suppressive organisms.
2. Development of a real time fluorescent PCR assay specific for Aphanomyces euteiches.
3. Determined the genotypic diversity of A. euteiches populations in field soils.
4. Measured the distribution and stability of soils naturally suppressive to Phytopthora cinnamomi.
5. Discovered several new organisms whose populations correlate with suppressiveness.
6. Isolated two species of Pseudomonas - Pseudomans tolaasii and Pseudomonas costantinii that can control Phytophthora cinnamomi hyphae
7. Compared Rhizoctonia populations at different positions within the patch and at different soil depths. Determined that natural suppression may develop with monocropping of a susceptible crop.
8. Identified a family of Osmotin genes from potatoes associated with pathogen resistance.
9. Identified genes in a negative signaling bacterium that affect antibiotic production
10. . Identified a post-transcriptional regulatory system and a two component regulatory system that down regulate antibiotic production in a biocontrol agent.
11. Characterized several enzymatic steps in phenazine antibiotic production.
12. . Identified unique proteins in T. atroviride that were differentially expressed in association with B. cinerea, P. erythroseptica, P. infestans and R. solani.
13. Analyzed the species and complexes of Pythium species in the Pacific Northwest and are beginning to understand the range of virulence among species.
14. Developed a technique to accurately quantify Pythium damage (root scanning).
15. Determined that the application of mixtures of transgenic biocontrol strains caused only small and transient disturbances in the structure of bacterial communities that colonized the wheat roots.
16. Identified wheat genes induced and repressed genes by treatment with Q8r1.
17. Identified phage and bacteriocins genes that may play roles in fitness in the rhizosphere
18. Showed that Pseudomonas putida can significantly reduce the affects of avocado root rot.
19. Repeated application of low inoculum levels by Bio-Ject results in high populations of biocontrol agents in the field.
20. Green manures like Sudan grass suppress nematodes through the release of toxic products (primarily hydrogen cyanide) from the degradation of the cyanogenic glucoside (dhurrin) during decomposition of the green manure in soil.
21. Showed that different fields differ with respect to nematode susceptibility as a measure of soil health.
22. Developing post-harvest strategies for the control of the root-infecting fungus Monosporascus cannonballus.
23. Pseudomonas corrugata is being commercialized with industry cooperators based on the work of two patents (Chun, W. 2000. US Patent No. 6,156,560. Use of Pseudomonas corrugata as a biological control agent. December 5, 2000. Chun, W. 2002. US Patent No. 6,383,798. Method for selecting desiccation tolerant strains of bacteria. May 20, 2002.).
24. Field trails to control stolon decay in Peppermint have been initiated.
25. An economic analysis of the cost effectiveness of Brassica treatment for the control of soil-borne diseases is being conducted.
26. Completion of the genomic sequence of P. fluorescens Pf-5 will allow molecular analysis of the traits important for bacterial biological control.
27. The production of the antifungal secondary metabolite pyoluteorin is autoregulated

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Report Information

Participants

See attached minutes for list of meeting attendees.

Brief Summary of Minutes

Accomplishments

III. Principal Accomplishments by Objective:<br /> <br /> Objective 1: To identify and characterize plant microbe interactions that provide suppression of diseases caused by soilborne pathogens.<br /> <br /> California (UCR - Stanghellini) identified an entomopathogenic fungus (Beauveria bassiana) which rapidly kills shore flies and developed a novel method for the delivery of the entomopathogen in greenhouse cultivation systems. A chemical that selectively enhances the population of fluorescent pseudomonads in recycled irrigation water was identified. Enhancement of the population was correlated to control of root rot of diverse plants caused by species of Pythium and Phytophthora.<br /> <br /> California (UCR - Becker): Strains of D. oviparasitica, F. oxysporum and Zoogloea sp. have been identified as potentially involved in soil suppressiveness against H. schachtii, the beet cyst nematode. Re-introduction of D. oviparasitica transferred and established the suppressiveness to a similar degree as in the original suppressive soil. Three additional beet cyst nematode-suppressive soils were identified in a field survey of H. schachtii-infested sites. The identification of the causal factors in the soil suppressiveness at the UC Riverside field station will provide the basis for studies that address the introduction and management of suppressive factors in disturbed and non-disturbed environments. The survey indicated that suppressive soils could be found in commercial agricultural fields. However, their occurrence is rare and not obviously related to physical soil properties.<br /> <br /> California (UCR - Borneman) demonstrated the effectiveness of D. oviparasitica in controlling H. schachtii populations in both different soil types and field trials. These results bode well for the future development of new and more sustainable H. schachtii management strategies.<br /> <br /> California (UCR - Menge): Further progress has been made in isolating and identifying biocontrol agents of P. cinnamomi. The molecular identification of the microorganisms in the suppressive Somis soil, which is the culmination of four years worth of work, has begun and should be done in six months. Pseudomonas constantinii and P. tolaasii have been identified from New Guinea soil as excellent biocontrol agents of P. cinnamomii. Further evaluation of these organisms will begin pending receipt of USDA permits. Greenhouse evaluation of other potential biocontrol agents, including from commercial sources, has been unsuccessful but is ongoing.<br /> <br /> New York (Abawi): In collaboration with the soil health program team at Cornell, the root health status (general soil suppressive capacity) of soils collected from a large number of vegetable production sites statewide was determined by conducting a soil bioassay with beans. Results obtained demonstrated that significant differences in root health occur among these soils.<br /> <br /> Oregon (Parke): Phytophthora ramorum causes Sudden Oak Death and ramorum blight on many nursery crops. This quarantine pathogen is considered to be a strictly foliar pathogen. We discovered that P. ramorum in potting media can infect rhododendron roots, leading to disease of stems and leaves via vascular tissue. Current plant inspections and nursery certification are based solely on foliar samples. Recovery of P. ramorum from symptomless root tissue demonstrates the need to monitor belowground sources of inoculum. This may be necessary to limit transmission of P. ramorum from nursery plants to susceptible oaks and other native vegetation in the eastern U.S. Future work will address how P. ramorum survives in potting media and recirculating irrigation water and will determine if these will be amenable to biological control approaches.<br /> <br /> Washington (Paulitz): Rhizoctonia bare patch is caused by R. solani AG-8, and is a major problem in direct-seeded wheat and barley. Because of the wide host range of this pathogen, crop rotation had not been considered feasible as a cultural practice to manage this disease. Recent field trials have demonstrated that wheat following barley has less disease and greater yield than continuous wheat. Because barley is also a susceptible host, the rotation effect is not due to lack of a host, but may be due to a shift in microbial communities. This suppression has also been demonstrated in the greenhouse, with intact soil cores taken from the centers of the patches and planted with 5 cycles of barley over one year. At the start of the experiment, barley was stunted, but by the end of the experiment, the stunting was not visible in most patches. The pathogen was still present in the cores that had become suppressive. This suggests microbial suppression may be involved.<br /> Objective 2: To understand how biological and environmental factors regulate microbial populations and the expression of genes responsible for disease suppression.<br /> <br /> Alaska (McBeath): Trichoderma atroviride is a cold tolerant fungus that parasitizes a wide range of plant pathogenic fungi. T. atroviride is very robust. Results of compatibility studies show that T. atroviride growth is not adversely affected by high concentrations of heavy metals. T. atroviride was found efficacious against Armillaria root rot on fruit trees. No further dieback was found on trees treated with T. atroviride. Treated trees are significantly taller with thicker trunks, larger and greener leaves than the untreated control.<br /> <br /> b-N-acetyl-D-glucosaminidase, b-1,6-glucanases and endo-b-1,3-glucanase are important enzymes involved in biocontrol by T. atroviride. The 73 kDa b-N-acetyl-D-glucosaminidase was produced only when T. atroviride was grown with S. sclerotiorum. T. atroviride produced the 93 kDa b-N-acetyl-D-glucosaminidase when it was grown with other plant pathogenic fungi. b-1,6-glucanases produced by cold tolerant T. atroviride were influenced by carbon source and pH. The 77 kDa endo-b-1,3-glucanase showed strong antifungal activity by inhibiting spore germination of Botrytis cinerea. <br /> <br /> Glutathione S-transferases (GSTs) and late blight resistance (R1) genes were shown to be responsive to biotic and abiotic stresses. Among the 33 potato cultivars tested, 3 cultivars had an amplification product for R1, 3 had amplification products for R1 and GST and 3 cultivars had amplification products for R1 and GST which are linked.<br /> <br /> Arizona (Pierson): Characterization of the molecular mechanisms responsible for phenazine gene regulation in P. aureofaciens 30-84 is continuing to provide insights into how root-associated bacteria sense its environment and response to it by altering patterns of gene expression. Ecological approaches are being used to quantify the role of cross-communication among rhizobacteria on phenazine gene expression in situ.<br /> <br /> Oregon (Loper): The genomic sequence of Pseudomonas fluorescens Pf-5 was completed in 2003 at TIGR and manual annotation is nearly complete (http://www.ars-grin.gov/hcrl/Pf5genome/index.htm). The total lengths of known and putative gene clusters encoding for antibiotic or siderophore biosynthesis is estimated at ca. 400 kb, representing approximately 5.7% of the 7.1 Mb genome of Pf-5. Of the 6147 putative genes in the Pf-5 genome, 681 have no homologs in the genomes of other Pseudomonas whose genome sequences are known. Oligomers (70 mers) representing each gene in the Pf-5 genome were designed for high-density microarrays. Low-density oligonucleotide microarrays were used to evaluate the influence of PltR (a regulator of the pyoluteorin biosynthesis gene cluster) on expression of 81 genes by Pf-5 and results were confirmed with quantitative RT-PCR. The same methods will be employed in 2005 to evaluate patterns of gene expression using the high-density (18K feature) microarray of Pf-5.<br /> <br /> Oregon (Parke): Sequencing the genome of biological control strain Burkholderia cepacia AMMD will enable investigations of the molecular basis of biocontrol and the soil factors which affect gene expression.<br /> <br /> Washington (Paulitz) has developed species-specific primers for the molecular detection of 10 Pythium spp., 3 groups of Rhizoctonia oryzae, and 3 groups of R. solani. These methods will allow the quantification of these pathogens from plant and soil samples, and the study of pathogen ecology and the influence of biological control agents.<br /> <br /> <br /> Objective 3: To develop and implement economic biological control systems to achieve sustainable agriculture. <br /> <br /> New York (Abawi): The plant activators Actigard and BioYield were evaluated in experimental fields for their efficacy against R. solani and Meloidogyne hapla on table beets and onions, respectively. Both products reduced the severity of diseases caused by the target pathogens and increased yield as compared to the untreated checks. The efficacy of these and other products against soilborne pathogens of vegetable crops will be re-evaluated in 2005.<br /> <br /> Montana (Callan): Peppermint stolon decay is caused by a complex of soilborne pathogens, including Pythium, Rhizoctonia, Fusarium, and Sclerotinia. Replicated plots were established to evaluate several biocontrol fungi and bacteria for protection of peppermint and improve winter survival. Establishment of a vigorous peppermint stand, free of diseases, insects, and weeds, is essential to the success and profitability of the peppermint industry in Montana.<br /> <br /> <br /> IV. Work Planned for Next Year:<br /> <br /> The members of this project plan to continue and advance the research efforts currently in progress. Several states, including CA and NY, will continue evaluating soils for potential suppressiveness and will determine the causal agents and mechanisms. WA, AZ, OR, and AK will continue to elucidate genetic mechanisms involved in biocontrol. California will also conduct factorial trials with strains of D. oviparasitica, F. oxysporum and Zoogloea sp. to evaluate potential interactions in reducing the beet cyst nematode population. Pseudomonas constantinii and P. tolaasii will be tested for control of P. cinnamomi.<br /> <br /> Application of biocontrol technology will be studied in several states. Plant activators, available biological control preparations and other products will be evaluated against soilborne pathogens and their damage to crops in NY and MT. Oregon will determine how soil and potting media components affect the survival of P. ramorum, and begin investigations on the survival and biological control of this pathogen in recirculating irrigation water used in many nurseries. They will also determine if colonization of roots and vascular tissue occur in other susceptible host species. <br /> <br /> <br /> V. Collaboration with Other W-147 Members: <br /> <br /> The W-1147 committee members have traditionally been active in research collaboration. Mike Stanghellini (CA-UCR) obtained two Pseudomonas isolates from L. S. Pierson (AZ) for investigations relating to N-Serve- amended nutrient solutions and initiated collaborative studies with James Borneman on the identification of microbes that induce germination of ascospores of M. cannonballus. Ole Becker (CA-UCR) and John Menge (CA-UCR) are also collaborating with James Borneman and his lab. Jenifer McBeath (AK) has provided Trichoderma atroviride to Nancy Callan (MT) for peppermint stolon decay studies. Jennifer Parke (OR) will confer and collaborate with John Menge (CA-UCR) and Mike Stanghellini (CA-UCR) on approaches for biocontrol of Phytophthora species in potting media and recirculating irrigation water used in nursery crop production. Tim Paulitz (WA) has collaborated with Nik Grunwald (WA), looking at ecology of Pythium in native soils and has also discussed ideas about Phytophthora suppression and parallels with Rhizoctonia patch suppression with John Menge (CA-UCR) and his graduate students. The WA unit has a long collaboration with L. S. Pierson (AZ) in studying phenazine biosynthetic pathways.<br />

Publications

ALASKA<br /> <br /> Cheng, M., Gay, P. A, and McBeath, J. H. 2004. Enzyme profiles associated with a cold tolerant Trichoderma atroviride during low temperature biocontrol of pathogenic fungi. Phytopathology 94:S18.<br /> <br /> Cheng, M., Gay, P. A. and McBeath, J. H. 2004. Trichoderma atroviride strain biotype 603, ATCC 74018 73 kDa N-acetyl-D-glucosaminidase gene, partial cds. GenBank Accession Number: AY646221, National Center for Biotechnology Information, Bethesda, MD.<br /> <br /> Cheng, M., Gay, P. A. and McBeath, J. H. 2004. Trichoderma atroviride strain biotype 453, ATCC 74016 73 kDa N-acetyl-D-glucosaminidase gene, partial cds. GenBank Accession Number: AY612745, National Center for Biotechnology Information, Bethesda, MD.<br /> <br /> Cheng, M., Gay, P. A. and McBeath, J. H. 2004. Trichoderma atroviride strain biotype 861, ATCC 74015 73 kDa N-acetyl-D-glucosaminidase gene, partial cds. GenBank Accession Number: AY646222, National Center for Biotechnology Information, Bethesda, MD.<br /> <br /> Gay, P. A. and McBeath, J.H. 2004. Solanum tuberosum cultivar German Butterball late blight resistance gene, partial sequence. GenBank Accession Number: AY582921, National Center for Biotechnology Information, Bethesda, MS. <br /> <br /> McBeath, J. H., Cheng, M., Gay, P., Ma, M., and Alden, J. 2004. First report of Leptographium abietinum associated with blue stain on declining western Siberian larch in Alaska. Online. Plant Health Progress doi: 10. 1094/PHP-2004-0326-01-HN.<br /> <br /> Gay, P. A. and McBeath, J.H. 2004. Solanum tuberosum cultivar 24-3 late blight resistance gene, partial sequence. GenBank Accession Number: AY582922, National Center for Biotechnology Information, Bethesda, MS.<br /> <br /> Gay, P. A. and McBeath, J.H. 2004. Solanum tuberosum cultivar Yellow Finn late blight resistance gene, partial sequence. GenBank Accession Number: AY582923, National Center for Biotechnology Information, Bethesda, MS.<br /> <br /> Gay, P. A. and McBeath, J.H. 2004. Solanum tuberosum cultivar Reba glutathione S-transferase cold induced gene, partial sequence. GenBank Accession Number: AY582924, National Center for Biotechnology Information, Bethesda, MS.<br /> <br /> Gay, P. A. and McBeath, J.H. 2004. Solanum tuberosum cultivar 772 late blight resistance gene, partial sequence. GenBank Accession Number: AY582925, National Center for Biotechnology Information, Bethesda, MS.<br /> <br /> Gay, P. A. and McBeath, J.H. 2004. Solanum tuberosum cultivar Snow Chip late blight resistance and cold induced glutathione S-transferase genes, partial sequence. GenBank Accession Number: AY582926, National Center for Biotechnology Information, Bethesda, MS.<br /> <br /> Gay, P. A. and McBeath, J.H. 2004. Solanum tuberosum cultivar Iditared late blight resistance gene, partial sequence. GenBank Accession Number: AY582927, National Center for Biotechnology Information, Bethesda, MS.<br /> <br /> Gay, P.A., McBeath, J.H. and Yokogi, T. 2004. Urocystis agropyri large subunit ribosonal RNA gene, partial sequence. GenBank Accession Number: AY547283, National Center for Biotechnology Information, Bethesda, MS.<br /> <br /> ARIZONA<br /> <br /> Morello, JE, Pierson, EA, and LS Pierson III. 2004. Negative cross-communication among wheat rhizosphere bacteria: Effect on antibiotic production by the biological control bacterium Pseudomonas aureofaciens 30-84. Appl. Environ. Microbiol. 70:3103-3109.<br /> <br /> McLean RJ, Pierson LS III, Fuqua C. 2004. A simple screening protocol for the identification of quorum signal antagonists. J Microbiol Methods. 58:351-360.<br /> <br /> Wijeratne EM, Carbonezi CA, Takahashi JA, Seliga CJ, Turbyville TJ, Pierson EE, Pierson LS III, VanEtten HD, Whitesell L, Bolzani Vda S, Gunatilaka AA. 2004. Isolation, optimization of production and structure-activity relationship studies of monocillin I, the cytotoxic constituent of Paraphaeosphaeria quadriseptata. Antibiot (Tokyo).57:541-546.<br /> <br /> Zhan J, Wijeratne EM, Seliga CJ, Zhang J, Pierson EE, Pierson LS III, Vanetten HD, Gunatilaka AA. 2004. A new anthraquinone and cytotoxic curvularins of a Penicillium sp. from the rhizosphere of Fallugia paradoxa of the Sonoran desert. J Antibiot (Tokyo). 57:341-344.<br /> <br /> He, J, Wijeratne, EMK, Bashyal, BP, Zhan, J, Seliga, CJ, Liu, MX, Pierson, EA, Pierson, LS III, VanEtten, HD, Ginatilaka, AAL. 2004. Cytotoxic and other metabolites of Aspergillus inhabiting the rhizosphere of Sonoran Desert plants. J. Nat. Prod. (in press).<br /> <br /> CALIFORNIA<br /> <br /> Becker, J.O., and J. Borneman 2004. Decoding the nature of a nematode-suppressive soil. Phytopathology 94:S129 (Abstr.).<br /> <br /> Becker, J.O, and J. Borneman 2004. Analysis of biological nematode population control: a case study. California Conference on Biological Control IV (Ed. M.S. Hoddle), Berkeley, CA, 23-27.<br /> <br /> Borneman, J., R. Olatinwo, B. Yin, and J.O. Becker 2004. An experimental approach for identifying microorganisms involved in specified functions: utilization for understanding a nematode suppressive soil. Australasian Plant Pathology 33:151-155.<br /> <br /> Chen, X., and J.O. Becker 2004. Dactylella oviparasitica as biocontrol agent for Meloidogyne incognita in tomato. Proceedings 15th International Plant Protection Congress, Beijing, China May 11-16,2004, p. 158 (Abstr.).<br /> <br /> El-Hamalawi, Z.A., and Stanghellini, M.E. 2004. Disease development on lisianthus (Eustoma grandiflorum) following aerial transmission of Fusarium avenaceum by adult shore flies, fungus gnats, and moth flies. Phytopathology 94:S27.<br /> <br /> McDonald, V.T. and J.A. Menge. 2004. Microbial suppression of Phytophthora cinnamomi in avocado soils. Phytopathology 94:S68 (Abstr.).<br /> <br /> Menge, J.A. and McDonald, V.T. 2004. Management of soil microorganisms for the control of Phytophthora root rot. Phytopathology 94:S125 (Abstr.).<br /> <br /> Olatinwo, R.O., B. Yin, J.O. Becker, and J. Borneman. 2004. An approach for identifying microorganisms involved in soil suppressiveness: utilization for identifying microorganisms that suppress the plant-parasitic nematode, Heterodera schachtii. Phytopathology 94:S78 (Abstr.).<br /> <br /> Pagliaccia, D., Merhaut, D., and Stanghellini, M.E. 2004. Enhancement of the fluorescent pseudomonad population after amending the recirculating nutrient solution of hydroponically-grown plants with a nitrogen stabilizer. Phytopathology 94:S80.<br /> <br /> Radewald,K.C., Ferrin, D.M., and Stanghellini, M.E. 2004. Sanitation practices that inhibit reproduction of Monosporascus cannonballus in melon roots left in the field after crop termination. Plant Pathology 53:660-668.<br /> <br /> Stanghellini, M.E., Waugh, M.M., Radewald, K.C., Kim, D.H., Ferrin, D.M. and Turini, T. 2004. Crop residue destruction strategies that enhance rather than inhibit reproduction of Monosporascus cannonballus. Plant Pathology 53:50-53.<br /> <br /> Stanghellini, M.E., Kim, D.H., Waugh, M.M., Ferrin, D.M., Alcantara, T. and Rasmussen, S.L. 2004. Infection and colonization of melon roots by Monosporascus cannonballus in two cropping seasons in Arizona and California. Plant Pathology 53:54-57.<br /> <br /> Stanghellini, M.E., and El-Hamalawi, Z.A. 2004. Efficacy of Beauveria bassiana on colonized millet seed as a biopesticide for the control of shore flies in greenhouses. Phytopathology 94:S98.<br /> <br /> Yin, B.A., A.J. Scupham, J.A. Menge, and J. Borneman. 2004. Identifying microorganisms similar to that of the pathogen: a new investigative approach for discovering biocontrol organisms. Plant and Soil 259:19-27.<br /> <br /> <br /> MONTANA<br /> <br /> Yang, Y., Chang, K. F., Hwang, S. F., Callan, N. W., Howard, R. J., and Blade, S. F. 2004. Biological control of Pythium damping-off in Echinacea angustifolia with Trichoderma species. Zeitschrift fur Pflanzenkrankheiten and Pflanzenschutz 111:126-136.<br /> <br /> <br /> NEW YORK <br /> <br /> Abawi, G. S., J. W. Ludwig, and C. H. Petzoldt. 2004. Assessing root health by a soil bioassay with beans as an indicator of soil health. Phytopathology 94 (Suppl.): S1 (Abstr. #579B).<br /> <br /> Abawi, G. S. 2004. Soil; Health Program Work Team at Cornell. PP. 35-36. Proceedings III, Empire State Fruit & Vegetable Expo, CCE, Cornell University, Ithaca, NY.<br /> <br /> Abawi, G. S. and J. W. Ludwig. 2004. Beet diseases, management options and research results, 2003. PP. 106-108. Proceedings III, Empire State Fruit & Vegetable Expo, CCE, Cornell University, Ithaca, NY.<br /> <br /> <br /> OREGON<br /> <br /> Anderson, L.M., Stockwell, V.O., and Loper, J.E. 2004. An extracellular protease of Pseudomonas fluorescens Pf-5 inactivates antibiotics of Pantoea agglomerans. Phytopathology 94:1228-1234.<br /> <br /> Brodhagen, M., Henkels, M. D., and Loper, J. E. 2004. Positive autoregulation of the antibiotic pyoluteorin in the biological control organism Pseudomonas fluorescens Pf-5. Appl. Environ. Microbiol. 70:1758-1766.<br /> <br /> Hansen, E. M., Parke, J. L., and Sutton, W. 2004. Susceptibility of Oregon forest trees and shrubs to Phytophthora ramorum: a comparison of artificial inoculation and natural infection. Plant Dis. (in press).<br /> <br /> Lewis, C. D., Roth, M. L., Choquette, C. J. and Parke, J.L. 2004. Root infection of rhododendron by Phytophthora ramorum. Phytopathology 94:S60 (Abstr.)<br /> <br /> Parke, J., Frankel, S., Alexander, J. and Thomas, C. 2004. Phytophthora ramorum Educate to Detect (PRED) Program. http://www.ncipmc.org/sod/<br /> <br /> Parke, J. L., Linderman, R. G., Osterbauer, N. K., and Griesbach, J. A. 2004. Detection of Phytophthora ramorum blight in Oregon nurseries and completion of Kochs Postulates on Pieris, Rhododendron, Viburnum, and Camellia. Plant Dis. 88:87.<br /> <br /> Parke, J., Pscheidt, J., and Linderman, R. 2004. Phytophthora ramorum: a guide for Washington nurseries. 8 pp. Washington State Nursery and Landscape Association.<br /> <br /> Parke, J. L. and Roth, M. L. 2004. Relative virulence of Phytophthora ramorum isolates in Oregon. Phytopathology 94:S81 (Abstr.).<br /> <br /> Parke, J. L., Roth, M. L., Lewis, C. and Choquette, C.J. 2004. Infection of Rhododendron macrophyllum roots, stems, and leaves by soilborne inoculum of Phytophthora ramorum. Proceedings, IUFRO Conference on Phytophthora in Forests, Freising, Germany, Sept. 11-17.<br /> <br /> Temple, T., Stockwell, V.O., Loper, J.E., and Johnson, K.B. 2004. Bioavailability of iron to Pseudomonas fluorescens strain A506 on flowers of pear and apple. Phytopathology 94:1286-1294.<br /> <br /> WASHINGTON<br /> <br /> Ahuja, E. G., Mavrodi, D. V, Thomashow, L. S., and Blankenfeldt, W. 2004. Overexpression, purification and crystallization of PhzA, the first enzyme of the phenazine biosynthesis pathway of Pseudomonas fluorescens 2-79. Acta Crystallogr. D 60:1129-1131.<br /> <br /> Allende-Molar, R., Landa, B. B., and Weller, D. M. 2004. Long-term survival of different genotypes of 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas fluorescens in soil. Phytopathology 94:S4 (Abstr.).<br /> <br /> Blankenfeldt, W., Kuzin, A., Skarina, T., Korniyenko, Y., Tong, L., Bayer, P., Janning, P., Thomashow, L. S., and Mavrodi, D. V. 2004. Structure and function of the phenazine biosynthetic protein PhzF from Psesudomonas fluorescens. Proc. Natl. Acad. Sci. USA 101:16431-16436.<br /> <br /> Blouin Bankhead, S., Brown, A., Son, M. Y., Thomashow, L. S., and Weller, D. M. 2004. Population dynamics and non-target effects of transgenic Pseudomonas fluorescens in the rhizosphere of wheat and pea. Phytopathology 94:S8 (Abstr.).<br /> <br /> Blouin-Bankhead, S., Landa, B., Lutton, E., Weller, D. M., and McSpadden Gardener, B. B. 2004. Minimal changes in rhizobacterial population structure following root colonization by wild type and transgenic biocontrol strains. FEMS Microbiology Ecol. 49:307-318.<br /> <br /> De La Fuente, L., Landa, B. B., Thomashow, L. S., and Weller, D. M. 2004. Dynamics of rhizosphere competition among genotypes of 2,4-diacetylphlorogucinol (DAPG)-producing Pseudomonas fluorescens depends on the host crop. Phytopathology 94:S24 (Abstr.).<br /> <br /> De La Fuente, L., Thomashow, L. S., Weller, D. M., Bajsa, N., Quagliotto, L., Chernin L., and Arias, A. 2004. Pseudomonas fluorescens UP61 isolated from birdsfoot trefoil rhizosphere produces multiple antibiotics and exerts a broad spectrum of biocontrol activity. Eur. J. Plant Pathol. (in press).<br /> <br /> Higginbotham, R. W., Paulitz, T. C., and Kidwell, K. K. 2004. Virulence of Pythium species isolated from wheat fields in eastern Washington. Plant Dis. 88:1021-1026.<br /> <br /> Higginbotham, R. W., Paulitz, T. C., Campbell, K. G. and Kidwell, K. K. 2004. Evaluation of adapted wheat cultivars for tolerance to Pythium root rot. Plant Dis. 88:1027-1032.<br /> <br /> Huang, Z., Bonsall, R. F., Mavrodi, D. V., Weller, D. M., and Thomashow, L. S. 2004. Transformation of Pseudomonas fluorescens with genes for biosynthesis of phenazine-1-carboxylic acid improves biocontrol of Rhizoctonia root rot and in situ antibiotic production. FEMS Microbiol. Ecol. 49:243-251.<br /> <br /> Mavrodi, D. V., Bleimling, N., Thomashow, L. S., and Blankenfeldt, W. 2004. The purification, crystallization and preliminary structural characterization of PhzF, a key enzyme in the phenazine-biosynthesis pathway from Pseudomonas fluorescens 2-79. Acta Crystallogr. D 60:184-186.<br /> <br /> Mavrodi, D., Validov, S., Mavrodi, O., De La Fuente, L., Boronin, A., Weller, D., and Thomashow, L. 2004. Bacteriocin activity among 2,4-diacetylphloroglucinol (DAPG)-producing fluorescent Pseudomonas spp. Phytopathology 94:S67 (Abstr.).<br /> <br /> Mavrodi, O. V., Mavrodi, D. V., Weller, D. M., and Thomashow L. S. 2004. Role of sss recombinase and dsbA in root colonization by Pseudomonas fluorescens Q8r1-96. Phytopathology 94:S67 (Abstr.).<br /> <br /> Okubara, P., and Paulitz, T. C. 2004. Simultaneous identification and quantification of Rhizoctonia solani and R. oryzae from root samples using real-time PCR. Phytopathology 94:S77 (Abstr.).<br /> <br /> Okubara, P.A., and Skinner, D.Z. 2004. Microarray expression profiles of wheat roots and shoots during cold stress and root colonization by Pseudomonas fluorescens. Plant Biology 2004, Abstract No. 694 (Abstr.).<br /> <br /> Okubara, P.A., Kornoely, J.P., and Landa, B.B. 2004. Rhizosphere colonization of hexaploid wheat by Pseudomonas fluorescens strains Q8r1-96 and Q2-87 is cultivar-variable and associated with changes in gross root morphology. Biological Control, vol. 30, p. 392-403.<br /> <br /> Paulitz, T. C. and Rossi, R. E. 2004. Spatial distribution of Rhizoctonia solani and Rhizoctonia oryzae at three different scales in direct-seeded wheat. Can. J. Plant Path. 26:419 (Abstr.).<br /> <br /> Paulitz, T. C. And Schroeder, K. L. 2004. A method for the quantification of Rhizoctonia solani and Rhizoctonia oryzae from soil using toothpicks. Phytopathology 94:S82 (Abstr.).<br /> <br /> Paulitz, T. C., Dugan, F., Chen, W., and Grünwald, N. J. 2004. First report of Pythium irregulare on lentils in the United States. Plant Disease 88:310.<br /> <br /> Schroeder, K. L. and Paulitz, T. C. 2004. Real-time PCR identification of Pythium spp. from cereals in eastern Washington. Phytopathology 94:S94 (Abstr.).<br /> <br /> Thomashow, L. S., Delaney, S. M., Mavrodi, D. V., and Weller, D. M. 2004. Sequences encoding phzO and methods. United States Patent No. 6,737,260 B1 (Patent).<br /> <br /> Validov, S., Mavrodi, O., De La Fuente, L., Boronin, A., Weller, D., Thomashow, L., and Mavrodi, D. 2004. Antagonistic activity among 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas spp. FEMS Microbiol. Lett. (in press). <br /> <br /> Weller, D. M., van Pelt, J. A., Mavrodi, D. V., Pieterse, C. M. J., Bakker, P. A. H. M., and van Loon, L. C. 2004. Induced systemic resistance (ISR) in Arabidopsis against Pseudomonas syringae pv. tomato by 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas fluorescens. Phytopathology 94:S108 (Abstr.).<br />

Impact Statements

1. Research results offered an alternative means of Armillaria root rot control which is both safe and environmentally benign, and further elucidated the mechanism of hyperparasitism of biological control and disease resistance of potatoes.
2. Identification of the nature of soil suppressiveness will eventually provide us with the tools and the mechanisms of natural population control. Suppressive soils occur in commercial agricultural fields in Southern California. However, their occurrence is rare and not obviously related to physical soil properties.
3. Researchers demonstrated the effectiveness of D. oviparasitica in controlling H. schachtii populations in both different soil types and field trials, which bodes well for the development of new and more sustainable management strategies.
4. Searching for biocontrol agents at the location where the fungal pathogen, Phytophthora cinnamomi, originated has led to the discovery of many suppressive soils and several possible new biocontrol agents.
5. Use of the field fermentor for continuous application of biocontrol agents in irrigation water has proven to be an effective, safe, cost effective method of disseminating biocontrol agents.
6. Researchers developed a new method for the delivery of an entomopathogenic fungus for the control of shore flies in enclosed agricultural environments.
7. The use of biological control agents for control of peppermint stolon decay will result in reduced pesticide use by growers.
8. Identification of suppressive soils and assessing the impact of crop and soil management practices against soilborne pathogens will contribute to the development and implementation of soil IPM programs for root diseases.
9. Demonstrating the effectiveness of elicitors of inducible host resistance and other biological control products against soilborne pathogens will promote their use and the implementation of ecological crop production systems.
10. Researchers demonstrated that Phytophthora ramorum can be transmitted via infested potting media.
11. Researchers conducted six workshops about Phytophthora ramorum for Oregon nursery growers.
12. Researchers produced disease diagnostic brochures about Phytophthora ramorum for Oregon and Washington nursery growers, developed national training materials on P. ramorum and Sudden Oak Death for Master Gardeners and Extension staff, held a national teleconference reaching over 700 participants, and posted these downloadable instructional materials to the North Central IPM website http://www.ncipmc.org/sod/
13. The project contributes to efforts to improve agriculture by enhancing biologically-based pest management strategies. Knowledge of the genomic sequence of the biological control agent Pseudomonas fluorescens Pf-5 will pave the way for future research evaluating how biological control agents suppress plant diseases. Understanding biological control will promote the development of new ways to combat plant diseases without the use of pesticides.
14. New techniques have been developed for the detection and quantification of Pythium and Rhizoctonia, two groups of pathogens that are difficult to study using tradition soil plating.
15. The host plant and the bacterial genotype have a profound influence on the colonization of the roots and the competitiveness of strains.
16. Bacteriocins may play an ecological role in the competitiveness of Pseudomonas spp.
17. Wild type strains are more ecologically fit and competitive than transgenic strains that are constructed with a combination of antifungal compound biosynthetic pathways.

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Accomplishments

Objective 1: To identify and characterize plant microbe interactions that provide suppression of diseases caused by soilborne pathogens.<br /> <br /> California (UCR - Becker and Borneman): <br /> <br /> Worldwide, beet-cyst nematodes are major production constraints of beets and crucifers. We have investigated a field at the University of California Riverside Field Station that has been suppressive this nematode for about 25 years. Analyses of soils positively correlated with soil suppressiveness against H. schachtii have identified causal agents of the suppressiveness nature. In greenhouse trials, strains of D. oviparasitica and F. oxysporum were reintroduced into fumigated, non-suppressive soil. While F. oxysporum did not significantly decrease H. schachtii populations within two nematode generations, D. oviparasitica decreased the beet cyst nematode population to a similar level as in the suppressive soil. Moreover, the increased plant growth as a result of the pest reduction was similar to the non-infested check. This benefit of the biological control has not always been observed in the original suppressive soil. Microplot trials confirmed the greenhouse results but also indicated that Fusarium strains may require a longer time period to reduce nematode populations.<br /> <br /> California (UCR - Stanghellini): <br /> <br /> N-Serve studies: Initiate qualitative and quantitative assessment of changes in the resident bacterial, including the fluorescent pseudomonad, populations in recycled nutrient solutions amended with the chemical and determination of the biological effect of such changes on plant growth and disease control. Additionally, investigations will be initiated on the mechanism(s) associated with selective enhancement of specific bacterial populations in the chemical-amended and recycled nutrient solutions. These studies will involve the use of specific rifampicin-resistant strains of Pseudomonas spp. which have been previously associated with control of root- infecting pathogens or enhancing plant growth. Population densities of fluorescent Pseudomonas spp. increased from 103 CFU/ml to 106 CFU/ml within 48-72 hr after amending the recycled nutrient solution in a hydroponic cultural system with N-Serve. Similar results were obtained using xylene as the amendment. Pseudomonas putida, biotype A, was the dominant indigenous fluorescent Pseudomonas spp. identified following chemical amendment of the nutrient solution. In in vitro studies, significant population increases of known fluorescent Pseudomonas species (i.e., Pf-5 and P. aureofaciens 30-84) occurred following their addition to the nutrient solution in the presence of N-Serve but those populations were lower (2X) than the population increases of the indigenous fluorescent Pseudomonas spp. However, Pf-5 and P. aureofaciens 30-84 were not competitive with indigenous fluorescent Pseudomonas spp. in in vivo studies and their populations decreased significantly following their addition to the nutrient solution in the presence or absence of N-Serve. Significant disease control (using pepper plants as the host and Phytophthora capsici as the root pathogen in a model system) was achieved following amendment of the nutrient solution with various concentrations of N-Serve or xylene.<br /> <br /> Biosurfactants. Conclude studies on the efficacy of biosurfactants (Zonix and ThermX70) on the control of zoosporic pathogens in recycled nutrient solutions. Amending the nutrient solution with either a rhamnolipid or a saponin completely suppressed disease development (root rot of pepper caused by Phytophthora capsici) in both ebb-and-flow and top-irrigated cultural systems, with either an organic potting medium or rockwool. These results provide evidence that biosurfactants may be a suitable alternative to registered fungicides or synthetic surfactants in the management of diseases caused by zoosporic pathogens in cultural systems employing recycled irrigation water. <br /> <br /> New York (Cornell - Abawi, Harman, Smart): <br /> <br /> Suppressive capacity of vegetable soils under various soil management practices to soilborne pathogens: As part of a collaborative and multidisciplinary project conducted by the Cornell Soil Health Program Work Team, soil samples collected from various grower fields were assessed for their general suppressive capacity to soilborne pathogens (root health) using the soil bioassay with bean (described in detail later). In general, root health was improved with the implementation of reduced tillage, long rotations with grain crops, certain cover crops and organic amendments. Frequency distributions are being used to compare various management practices.<br /> <br /> Evaluation of selected cover crops incorporated as green manures for suppressing soilborne pathogens: Varieties of forage and grain pearl millets, canola, rapeseed, buckwheat, hairy vetch, sudangrass, oilseed radish and mustard are being evaluated in field microplots, an experimental root rot field and in the greenhouse against bean root rot pathogens, especially Rhizoctonia solani and Pratylenchus penetrans. A total of 18 replicated treatments were established in the field microplot (4-ft. diam. fiberglass cylinders) test. The field soil in the microplots was additionally infested with R. solani (potato-soil inoculum preparation) prior to planting with the various cover crops. After approximately 8 weeks, the cover crops were cut and incorporated into the soil. A month later, a soil sample was collected from each microplot and bioassayed with bean in the greenhouse. Root rot severity was rated 5 weeks later on a scale of 1 (healthy root) to 9 (>75% of roots and stem tissues affected and at various stages of decay). Results obtained suggest a differential effect of the various cover crops and varieties tested, even after being incorporated for only one month. The microplots will be planted to beans in spring 2006 and root rot severity and yield will be determined. Seven cover crop treatments were established in an experimental bean root rot field (heavily infested with R. solani, Thielaviopsis bascicola, Pythium ultimum, and Fusarium solani f. sp. phaseoli) in early August after the 2005 bean crop was plowed under. Each plot was 25 x 60 ft. and the treatments were replicated 4 times. The cover crops were incorporated in early October and soil samples were collected in early November for the greenhouse soil bioassay, which is still in progress. These plots will also be planted to beans in spring of 2006 for root rot and yield assessment and evaluation. An extensive greenhouse evaluation of all the selected cover crops and varieties is in progress and is been conducted in naturally infested soil and in soil infested with P. penetrans. <br /> <br /> Oregon (Oregon State - Parke): <br /> <br /> We are investigating the ecology and epidemiology of Phytophthora ramorum in soil, potting media, and irrigation water so as to design effective cultural and biological control measures for eliminating this disease in horticultural nurseries. Infection of roots and spread in vascular tissue: Phytophthora ramorum, cause of sudden oak death and ramorum blight, is believed to be an aerial plant pathogen. We demonstrated that P. ramorum can infect roots of rhododendron grown in artificially infested potting media, spreading in the vascular tissue to stems and leaf petioles. Foliar infections also spread into stem tissue via the vascular system. Survival in soil: P. ramorum was recovered up to 20 weeks after burial of detached inoculated rhododendron leaves in potting soil and forest soil moistened to achieve a range of soil water matric potentials (-1 to -1500 kPa). Taken together, results from our experiments indicate that P. ramorum has a soil-borne phase which could be important in the epidemiology of this disease, particularly in nurseries or in landscaped settings. The significance of the soil phase of P. ramorum in natural forest settings is not yet clear. Epidemiology and control of Phytophthora species in recirculating irrigation water: We initiated studies on the detection, epidemiology, and control of Phytophthora species in recirculating irrigation systems. We constructed 32 self-contained mini-nurseries, each with its own recirculating water system, for conducting dose-response studies and for testing the efficacy of water treatment methods, including biofilters, for the elimination of Phytophthora species in irrigation water.<br /> <br /> Washington (USDA - Paulitz, Okubara, Weller, Thomashow): <br /> <br /> Suppression of Rhizoctonia bare patch with barley rotations. Rhizoctonia bare patch is caused by R. solani AG-8, and is a major problem in direct-seeded wheat and barley. Because of the wide host range of this pathogen, crop rotation had not been considered feasible as a cultural practice to manage this disease. However, recent field trials have demonstrated that wheat following barley has less disease and greater yield than continuous wheat. Because barley is also a susceptible host, the rotation effect is not due to lack of a host, but may be due to a shift in microbial communities. This phenomenon was documented in the field again this year, and a manuscript was accepted in Plant Disease. <br /> <br /> Reduction of soilborne pathogens under long-term no-till. During the conversion from conventional tillage to no-till or direct seeding, certain root diseases can become more severe. This was documented in a four-year field trial near Pullman. No-till plots were established on land that had been conventionally tilled for a long period of time. Other plots at the site were kept as conventionally-tilled, and the experiment was planted with a spring wheat-spring barley rotation. Another location, about 1 km away, had been in no-till for 12 years, and both conventional and no-till plots were established. Rhizoctonia, Pythium, and take-all were monitored over the next four years, along with plant measurements. For the first 2 years, there was no effect of stopping tillage and converting to no-till. However, in the 3rd and 4th year of the conversion, Rhizoctonia became severe, and significantly reduced yield. Population studies documented the build-up of R. solani, but not R. oryae. However, converting the no-till plots back to tillage did not result in any increase of yield or decrease in disease. Another part of the study compared long-term no-till farms to adjacent conventionally-tilled farms. No differences were found in root diseases. This indicates that Rhizoctonia becomes severe a few years after tillage is stopped, but when no-till has been practiced for 12 years or longer, disease appears to be suppressed, compared to earlier years. This phenomenon has also been documented in Australia. Future research will focus on documenting the shift in microbial communities that accompanies the transition from conventional tillage to no-till, and compare long-term no-till fields to adjacent conventionally tilled fields.<br /> <br /> Objective 2: To understand how biological and environmental factors regulate microbial populations and the expression of genes responsible for disease suppression. <br /> <br /> Arizona (U of Arizona - Pierson): <br /> <br /> Molecular analysis of negative signaling among wheat rhizobacteria. We continue to analyze the negative signal(s) produced by the wheat rhizosphere strain PU-43. The negative signal produced by this strain blocks phenazine gene expression in strain 30-84 and b-galactosidase activity in the genomic reporter strain 30-84Z. It has proven difficult to purify the negative signal. A genomic library has been constructed from strain PU-43. We identified a cosmid that contains an AHL synthase gene that rescues an AHL- strain of 30-84. A knockout mutant has been constructed that only produces the negative signal. Currently the signal can be extracted into butanol that is allowing further structural characterization. We also have identified four other cosmids from this library that result in a reduction in phenazine gene expression and may encode negative signals. One of these cosmids contains the gene oprF. OprF is a major outer membrane-associated protein in pseudomonads. In the human pathogen P. aeruginosa, OprF was shown to bind human interferon-g (IFN-g) resulting in QS-dependent induction of the lectin PA-I and the phenazine pyocyanin. <br /> <br /> Characterization of RpeB, the second part of the two component regulatory system that negatively regulates phenazine production. RpeA is a sensor kinase that appears to couple aspects of cell metabolism to secondary metabolite production. Mutations in rpeA result in higher levels of phenazine gene expression in minimal medium in comparison to the wild type strain. We have cloned and sequenced the second component, rpeB of this two compontnet system. It is similar to other known two component response regulatory genes. Of great interest is the fact that 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 medium. 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.<br /> <br /> Identification of a new regulatory gene. We have identified a cosmid from the 30-84 genomic library that when present in trans in strain 30-84Ice with a second plasmid containing the phenazine promoter fused to lacZ reduces b-galactosidase expression 100-fold (2,000 vs. 20 Miller units). Localization of the region responsible implicates psrA as the gene responsible for the observed effect. Further analysis is ongoing.<br /> <br /> New York (Cornell - Abawi, Harman, Smart): <br /> <br /> Utility of plant growth-promoting rhizobacteria for disease control of foliar pathogens: In addition to having a direct effect on soilborne pathogens, some soil-inhabiting microbes provide disease control via a process known as induced systemic resistance (ISR). The role ISR may play in control of foliar pathogens is unclear. To gain a better understanding of how ISR may lead to disease control, we are following the gene expression patterns of plants that have either been treated with plant growth-promoting Bacillus sp. or left untreated. Expression of multiple genes has been followed both pre-and post inoculation with a bacterial pathogen. For several pathogenesis related genes, we have found an increase in gene expression only visible in tomatoes treated with ISR-inducing Bacillus spp. These results support the idea that plants treated with ISR-inducing products are primed and can respond more quickly upon pathogen attack. Although the ISR-induced plants show a quicker up-regulation of defense-response genes, there was no significant disease control on these plants. <br /> <br /> Oregon (USDA - Loper): <br /> <br /> The recently-published genome of Pseudomonas fluorescens is being exploited to study patterns of gene expression by this biological control agent. A limited number of microarray slides have been spotted and tested for use in gene expression studies. Methods for isolation of RNA have been adapted for Pf-5. <br /> <br /> We completed studies demonstrating a role for the stationary phase sigma factor (RpoS) in stress response and rhizosphere colonization by P. fluorescens Pf-5. In this bacterium, RpoS is required for optimial stress response, including its capacity to survive desiccation in culture and in the rhizosphere of plants grown in dry soils. This work provides the first evidence for a role of RpoS in environmental fitness of a bacterium in the field. <br /> <br /> Washington (USDA - Paulitz, Okubara, Weller, Thomashow): <br /> <br /> Development of species-specific PCR primers for the detection of Pythium spp. in wheat production. In order to understand how Pythium communities vary with rotation, season, tillage and other cultural practices, we need a method for accurately following the populations. Soil dilution plating cannot distinguish among the more that 10 species of Pythium that surveys have detected. We had developed and tested 10 primer sets for quantitative capillary real-time PCR, based on ITS sequences, that are specific for the following species- P. abappressorium, P. attrantheridium ( intermedium), P. irregulare IV, P. irregulare, P. ultimum, P. rostratum (rostratifingens), P. heterothallicum, P. sylvaticum, P. paroecandrum, and an unidentified species. We have developed standard curves and extracted DNA from natural soil to quantify Pythium. Studies were conducted this year to compare the Pythium species composition in different crop rotations. Seven Pythium species were detected, but preliminary data does not show any species shift among rotations during the beginning of the season, but, in general, populations decline over the growing season.<br /> <br /> Simultaneous identification and quantification of Rhizoctonia solani and R. oryzae using real-time PCR. Rhizoctonia is also difficult to quantify in soil, and cannot be detected with soil dilution plating because of the low population density. We have developed a semi-quantitative method using toothpick baiting, but we are also need a more accurate method that not only measures hyphal activity, but also dormant inoculum. Using ITS sequences, we have developed real-time PCR assays for 2 groups of R. oryzae. We have documented the presence of R. solani AG-8, AG-10, and AG-2-1, along with a binucleate Ceratobasidium spp. R. solani AG-2-1 is highly pathogenic on canola and also causes stunting of pea, but is not virulent on wheat or barley. <br /> <br /> Interactions between wheat cultivar and bacterial isolate in rhizosphere colonization and accumulation of 2,4-diacetylphlorogucinol (DAPG) produced by Pseudomonas fluorescens. Continuing studies have shown that wheat cultivars vary in their ability to support colonization by strains of P. fluorescens. A soil-free system was developed to assay DAPG production in roots and to do microarray studies. Cultivars varied in their ability to accumulate DAPG in roots. Some host defense/stress related genes were induced in wheat roots during colonization by P. fluorescens.<br /> <br /> Populations of DAPG-producing P. fluorescens in the rhizosphere on Pythium-infected wheat. Infections of roots by soilborne pathogens can either positively or negatively affect root colonization and antibiotic production by beneficial bacteria. We tested the impact of four Pythium spp. on colonization of wheat roots by indigenous populations of 2,4-diacetylphloroglucinol (DAPG)-producing P. fluorescens in Washington State suppressive soils. Before infestation with Pythium spp., soils were cycled to wheat (cv Penawawa) to reactivate and increase indigenous populations of the DAPG producers to a density greater than 105 CFU/g fresh root. Soils were then infested with P. abappressorium, P. irregulare, P. irregulare group IV, or P. ultimum at a rate of 103 CFU/g soil. In addition, 0.5% ground rolled oats was added to infested soils. Seven days after amending the soil, pregerminated wheat seed was sown. Rhizosphere populations of DAPG-producing bacteria were assessed using the PCR-based dilution end point assay 2 weeks after sowing. Populations of DAPG-producing P. fluorescens were similar in both infested and non-infested soils. None of the Pythium spp. affected the populations of indigenous DAPG-producing P. fluorescens on the wheat roots.<br /> <br /> Objective 3: To develop and implement economic biological control systems to achieve sustainable agriculture. <br /> <br /> Alaska (U of Alaska - McBeath): <br /> <br /> Efficacy of Trichoderma atroviride in the control of Armillaria mellea on plum trees in California (This study was conducted in collaboration with Ms. Kelly McBee Walker, AmPac Biotech, Fresno California and Mr. Jay Scott, Reedly Farm, Sanger, CA). Armillaria root rot is one of the most serious diseases on fruit trees. Methyl bromide soil fumigation has been the primary strategy for disease control. International restrictions on methyl bromide use make it necessary to seek alternatives. In mid-December, 2003, an efficacy study of Trichoderma atroviride was initiated in Sanger, CA on a plum orchard heavily infested with Armillaria root rot. A section of the farm, approximately 100 acres, was prepared as the study site. Plant Helperâ (flowable formulation), containing 107 cfu/g of T. atroviride spores, was sprayed on the root of plum stocks, at the application rate of one pound per acre, before being transplanted. Two rows of the transplants adjacent to the road were kept as an untreated control. Observations were made on the health of the trees. At the end of the second year, ten trees were randomly selected from the treated plot and untreated control and measured. The circumference of the tree trunk was taken at 12 inches above the crown. Data on leaf samples, ten leaves randomly harvested per tree, from the control and treated sample trees, were also collected and analyzed (using the Student t-test). The Plant Helperâ treatment continued to produce favorable results. The trunk of the treated trees, with a mean circumference of 32.88 cm was found significantly thicker than the untreated control (27.0 cm). Leaves from the treated trees were found to be significantly longer (14.35 cm) and broader (3.73 cm) than the untreated control (12.91 x 3.16 cm). Furthermore, the color of the leaves of the treated trees was greener as the leaves of the untreated control were in various stages of senescence.<br /> Growth and yield responses of potatoes to Trichoderma atroviride seed piece treatment in Alaska. Trichoderma atroviride is an extremely versatile microorganism. It has been found to parasitize and destroy a wide range of economically important plant pathogens, and sequestrate heavy metals. In 2005, results of an experiment conducted in a commercial potato field near North Pole, AK, illustrated the ability of T. atroviride to strongly enhance the growth and yield of plants. Plant Helperâ (dust formulation), containing 103 cfu/g of T. atroviride spores, was applied on cut potato seed pieces, at a rate of 15 kg of Plant Helperâ/400 kg of seed pieces/acre. A strip of the field was planted with untreated potato seed pieces as control. Potatoes were planted in early June. The first sampling taken in mid July, showed that the potato plants grown from treated seed pieces were more advanced in their development than the untreated control. In mid-August, ten plants each were randomly harvested for evaluation. Data collected were: numbers of main shoots, numbers of side shoots, plant weight (fresh and dry), root weight (fresh, dry), numbers of stolon, numbers and total weight of progeny potato tubers, numbers of marketable potatoes (>0.2 kg). Progeny tubers were also examined for any signs of scab, dry rot, or black scurf. Data was analyzed using the Student t-test. No lesion or sign of diseases was found on any of the progeny tubers examined. Significant difference was found between T. atroviride treated potatoes and untreated control on fresh plant weigh, dry plant weigh, fresh root weigh, dry root weigh, numbers of main shoots, numbers of side shoots, total weigh of progeny potato tubers, total numbers and weigh of marketable potatoes. No significant difference between T. atroviride treated potato and control on the numbers of stolon and progeny potato tubers/plant. However, treated potatoes had more main shoots, side shoots, biomass, root mass, larger potatoes and more marketable potato tubers. This experiment illustrated that T. atroviride promotes the growth and development of potato plants and increases the yield. T. atroviride seed piece treatment will enable farmers to harvest their potatoes one or two weeks earlier. In Alaska and northern countries where the growing season is short and weather capricious, T. atroviride treatment of seed pieces can be most beneficial to potato farmers. <br /> <br /> New York (Cornell - Abawi, Harman, Smart): <br /> <br /> Second year evaluation of plant activators and a biological nematicide for the control of soilborne pathogens on table beets, onions and cabbage. In 2004, foliar applications of Actigard (acibenzolar-S-methyl) showed promising results in controlling foliar infections caused by Rhizoctonia solani on table beets and the root-knot nematode (Meloidogyne hapla) on onion. However, Actigard applications in 2005 did not reduce the incidence of R. solani infections on beets or significantly affect marketable beet yield. Actigard applications to onions slightly reduced the root-galling severity and root-knot nematode numbers in roots and soil, but it was not significantly different from the control. Also, Actigard and the other control products included in the test had no significant effect on onion yield, probably due to the extreme dry and hot weather conditions that prevailed during the season. The inclusion of a biological nematicide (STAN, a product of Syngenta) as a component of a seed treatment package was effective in reducing the number of P. penetrans in roots and soil of cabbage seedlings in a growth chamber and also in field trials (data not shown). <br /> <br /> Screening of selected bean germplasm for resistance to root rot pathogens. In 2004 and 2005 replicated trials were conducted to evaluate the field reaction and yield response of a large number of bean germplasm lines and varieties grown under severe root rot pressure in an established root rot field at NYSAES in Geneva, NY. The germplasm lines included in these trials differed greatly in the percent of emergence, number of productive plants at harvest, root rot severity ratings and seed or pod yield. <br /> <br /> Field trials of plant growth-promoting rhizobacteria for the control of a bacterial pathogen of tomato. We have conducted a preliminary field experiment investigating the efficacy of systemic acquired resistance (SAR) and induced systemic resistance (ISR) inducers to control bacterial speck in the field in New York. The results of this experiment reveal that the SAR-inducing compound (acibenzolar-S-methyl) controlled the pathogen to the same level as the standard copper-based spray program. These results are similar to those observed in other states. However, the ISR-inducing Bacillus spp. was ineffective in disease control. The ISR-inducing bacteria did increase yield as measured by both fruit number and weight. Interestingly, when the SAR- and ISR-inducing products were combined, disease control was observed but there was no increase in yield. <br /> <br /> Montana (Montana State - Callan): <br /> <br /> Stolon Decay of Peppermint. Fall planting of peppermint in the Flathead Valley of Montana typically results in poor emergence the following spring. The resulting loss of stand leads to lower oil production and a greater requirement for weed control measures. Rhizoctonia, Fusarium, Sclerotinia, and Pythium have been isolated from stolon decay lesions. Research at Montana State Universitys Northwestern Agricultural Research Center (NWARC) has demonstrated the effectiveness of fungicide application at fall planting of mint stolons. <br /> <br /> Replicated plots were established at the NWARC on October 27, 2004, to evaluate fungicides and biocontrol agents for control of mint stolon decay. Plots consisted of four rows 4.6 m long and 0.3 m apart, with four replications. Stolon segments (1.4 kg, 10-15 cm long) were coated with 300 ml of the designated treatment and laid in furrows 4-5 cm deep. The viability of biological inocula was confirmed by dilution plating. <br /> <br /> Only the fungicides Gem and Prevail increased emerged plant stand in the spring of 2005 over the water-only control, and Gem, Tilt and Tops MZ increased dry matter production over the control. Oil yield was not significantly influenced by treatment, but was highly correlated with dry matter (r2 = 0.89, P = <0.0001). The relationship between stand and dry matter (r2 = 0.25, P = 0.0807) and stand and oil yield (r2 = 0.29, P = 0.0565) was weaker due to the ease of spread of the mint plant from stolons. <br /> <br /> Gem and Prevail, fungicides that were most effective in improving plant stand, are primarily labeled for control of Rhizoctonia diseases. Tops MZ and Tilt are also labeled for this pathogen. Tops MZ and Prevail increased dry matter and/or oil yield in previous studies of stolon decay in Montana. We can speculate that Rhizoctonia had a greater impact in this planting than did Pythium, Fusarium, and Sclerotinia, other genera of pathogenic fungi that have been isolated from decayed mint stolons. <br /> <br /> The biocontrol agents Trichoderma atroviride, Trichoderma harzianum, and Bacillus pumilus, did not increase stand and yield of peppermint under these conditions. Trichodeerma harzianum is labeled for Rhizoctonia diseases and B. pumilus has activity against Rhizoctonia solani (Kanjanamaneesathian, World J Microbiol Technol 16:523, 2000). Trichoderma atroviride is also effective against Rhizoctonia (McBeath, personal communication). <br /> <br /> <br /> <br /> V. Collaboration with Other W-147 Members: <br /> <br /> The USDA Pullman group will be initiating a collaboration with Borneman on how cultural methods affect microbial populations in no-till cereal production, and how a suppressive microflora may develop during long-term no-till. Our unit has a long collaboration with S. Pierson at Univ. of Arizona in studying phenazine biosynthetic pathways. Our unit has also collaborated with N. Grünwald on the development of AFLP methods to study populations of Rhizoctonia. Borneman and Stanghellini have a collaboration to identify microorganisms involved in the germination of Monosporascus cannonballus ascospores. Borneman and Becker have a collaboration to identify microorganisms involved in (i) Meloidogyne suppressiveness and (ii) replant suppression of walnut and peach. Determine the amount of Dactylella oviparasitica needed to create Heterodera schachtii suppressiveness and how crop type influences this suppressiveness. Loper and Pierson collaborate on studies evaluating gene expression of Pf-5 using microarrays. McBeath collaborates with Callan and Miller of Montana State on the control of decline of peppermint and snow mold of turfgrasses using Trichoderma. McBeath also collaborates with Mr. Jay Scott, Reedly Farm, Sanger, CA and Ms. K. Walker, AmPac Biotech Corporation, Fresno CA. Dr. Bowen McBeath, Portland State examining the control of Armillaria root rot on plum. <br />

Publications

Impact Statements

1. Identification of suppressive soils and assessing the impact of crop and soil management practices against soilborne pathogens will contribute to the development and implementation of soil IPM programs for root diseases.
2. Demonstrating the effectiveness of elicitors of inducible host resistance and other biological control products against soilborne pathogens will promote their use and the implementation of ecological crop production systems.
3. We discovered that P. ramorum can infect roots of rhododendron
4. We showed that P. ramorum can spread via the vascular system
5. We showed that P. ramorum can survive in soil and potting medium for at least 5 months
6. New techniques have been developed for the detection and quantification of Pythium and Rhizoctonia, two groups of pathogens that are difficult to study using tradition soil plating.
7. The host plant and the bacterial genotype have a profound influence on the colonization of the roots and the competitiveness of strains.
8. Colonization of wheat roots with Pythium spp. did not affect populations of indigenous DAPG producers.
9. Bacteriocins may play an ecological role in the competitiveness of Pseudomonas spp.
10. Newly discovered Type III secretion system genes in PGPR Pseudomonas spp. may play an important role in bacterial-plant interactions and biocontrol.
11. The effectiveness of D. oviparasitica in controlling H. schachtii populations in both different soil types and field trials bodes well for the development of new and more sustainable management strategies. These results suggest the likelihood of success of the newly initiated studies to identify microorganisms involved in Monosporascus cannonballus germination, Meloidogyne suppressiveness, peach and walnut replant suppression, and Pierces disease suppression are high.
12. Our research results on cold tolerant Trichoderma atroviride elucidated the mechanism of hyperparasitism of biological control, offered a safe and environmentally benign alternative means of Armillaria root rot control and a seed piece treatment which promotes plant growth, development and increase yield of potatoes.
13. Fungicidal treatments for control of peppermint stolon decay have been identified in replicated trials at the Northwestern Agricultural Research Center, Kalispell, MT.
14. We deposited the full genome sequence of the biological control agent Pseudomonas fluorescens Pf-5 in public databases for free access by the scientific community.
15. Identification of regulatory networks will allow manipulation of patterns of gene expression to increase pathogen inhibition.
16. Understanding the ecological inputs influencing mechanisms involved in pathogen inhibition will allow improved activity in complex microbial communities.
17. Understanding multiple roles of secondary metabolites will allow improved efficacy under field conditions.

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Accomplishments

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. <br /> <br /> California (UCR - Borneman): <br /> <br /> 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. <br /> <br /> 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.<br /> <br /> California (UCR - Stanghellini): <br /> <br /> 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. <br /> <br /> 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<br /> <br /> 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. <br /> <br /> Nebraska (Univ. of Nebraska- Gary Yuen)<br /> <br /> 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. <br /> <br /> New York (Cornell- Christine Smart and George Abawi)<br /> <br /> A. Assessing and documenting the suppressive capacity of the soil under different production systems:<br /> <br /> 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.<br /> <br /> 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. <br /> <br /> Ohio (Ohio State- Brian McSpadden-Gardener)<br /> <br /> 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. <br /> <br /> Oregon (Oregon State- Parke and Grunwald)<br /> <br /> 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.<br /> <br /> Washington (USDA-ARS, Pullman, Paulitz, Okubara, and Weller)<br /> <br /> 1. Pseudomonas spp. that produce phloroglucinol and are responsible for take-all suppression in wheat, do not suppress Pythium root rot.<br /> <br /> To assess the role of DAPG producers in the suppression of take-all, soils that had<br /> 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<br /> inhibited in vitro mycelial growth of Pythium spp.; however, TAD soils from Lind and<br /> Quincy, harboring large populations of indigenous DAPG-producing P. fluorescens, were<br /> not suppressive to Pythium root rot. Pythium root rot severity was similar on<br /> Penawawa wheat seedlings grown in soils conducive or suppressive to take-all infested<br /> 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<br /> experiments, wheat seeds treated with different DAPG-producing P. fluorescens<br /> genotypes or the recombinant strains Z30-97 or Z34-97, able to produce phenazine-1-<br /> carboxylic acid and DAPG, did not reduce Pythium root rot on wheat seedlings.<br /> <br /> 2. Fallow from mechanical tillage or herbicide results in transient reduction in Rhizoctonia activity.<br /> 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.<br /> <br /> 3. Composition of Pythium spp. in cereal cropping systems is influenced by crop rotations and fallow<br /> 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. <br /> <br /> <br /> 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.<br /> <br /> Arizona (Univ. of Arizona- Pierson):<br /> <br /> 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. <br /> 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. <br /> <br /> 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. <br /> <br /> 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.<br /> <br /> 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.<br /> <br /> Nebraska (Univ. of Nebraska- Gary Yuen)<br /> <br /> 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. <br /> <br /> New York (Cornell- Christine Smart and George Abawi)<br /> <br /> A. Evaluation of a biological nematicide for use as a seed treatment against northern root-knot nematode on vegetables:<br /> <br /> 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.<br /> <br /> 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.<br /> <br /> B. Utility of plant growth-promoting rhizobacteria for disease control of foliar pathogens:<br /> <br /> 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. <br /> <br /> Ohio (Ohio State- Brian McSpadden-Gardener)<br /> <br /> 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. <br /> <br /> Oregon (USDA-ARS, Corvallis, Loper)<br /> <br /> 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. <br /> <br /> Washington (USDA-ARS, Pullman, Paulitz, Okubara, and Weller)<br /> <br /> 1. Development of real-time PCR for quantification of DAPG genotypes of Pseudomonas fluorescens.<br /> <br /> 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. <br /> <br /> 2. Effect of Pseudomonas fluorescens isolate Q8r1-96 on gene expression in wheat roots<br /> <br /> 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<br /> <br /> Objective 3 To develop and implement biological control in agriculture. <br /> <br /> New York (Cornell- Christine Smart and George Abawi)<br /> <br /> 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.<br /> <br /> Ohio (Ohio State- Brian McSpadden-Gardener)<br /> <br /> 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.<br /> <br /> V. Collaborations with other W-1147 members<br /> <br /> 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.

Publications

Impact Statements

1. These studies are to identify microorganisms involved in Meloidogyne suppressiveness as well as peach and walnut replant suppression, which should lead to the development of new and more sustainable strategies to manage these pests/diseases.
2. Identification of regulatory networks with Pseudomonas will allow manipulation of patterns of gene expression to increase pathogen inhibition.
3. Understanding the ecological inputs influencing mechanisms involved in pathogen inhibition will allow improved activity in complex microbial communities.
4. Understanding multiple roles of secondary metabolites of Pseudomonas will allow improved efficacy under field conditions.
5. Research with Lysobacter enzymogenes may lead to the development of biocontrol agents for fungi and nematodes destructive to plants, thus reducing loss from plant diseases while reducing the input of chemical pesticides to the environment.
6. Research at Ohio State has identified organic transition practices that can significantly reduce soilborne disease pressure in multiple crops.
7. The lab of Dr. McSpadden-Gardener has developed and tested novel biological seed treatments for enhanced productivity on conventional and organic farms and has developed a convenient, stable, and low cost formulation for on-farm bacterial seed treatments.
8. It is critical that the management of soilborne pathogens and their resultant root diseases to be a compatible component of overall soil health management practices. Thus, the results in New York have been collected on the effects of promoted soil health management practices on root pathogens and their damage will contribute to development of appropriate control options that are ecologically based and also promoting soil health in general.
9. For biological control agents to be implemented, it is necessary to understand the impact they have on induction of plant defense responses. The only way to determine this is to study gene expression of marker genes from plants grown in the field using typical agricultural practices. This project in New York will identify when plant defense responses are activated, which will allow for the best possible timing of application of biological control products to reduce disease incidence.
10. Parke (OR) has discovered that P. ramorum can infect roots of tanoak under natural conditions in the forest.
11. The group at Oregon State initiated a multi-year study to develop a new approach for identifying and eliminating sources of contamination by Phytophthora species in nurseries through targeted changes in cultural practices.
12. Although DAPG producers have been implicated in suppression of take-all caused by Gaeumannomyces graminis var. tritici, Pythium spp. in wheat are not as sensitive to this antifungal compound and root rot may not be reduced. However, it is not known what effect these bacteria may have on the protection of wheat seed from Pythium.
13. Multiple species of Pythium and Rhizoctonia can be quantified in soil using PCR technology. This will lead to better diagnositics.
14. Cropping systems (crop rotation and fallow) affect species of Pythium, both abundance and population density.
15. Pseudomonas fluorescens induces higher expression of genes in wheat roots that are involved in disease defense.
16. Different phlD genotypes of Pseudomonas fluorescens can be quantified in mixtures in natural soil with real-time PCR.

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Alaska (Univ of Alaska - McBeath)<br /> Plant Helperâ, a formulated Trichoderma atroviride, continues to demonstrate that it is efficacious in the control of Armillaria root rot of fruit trees. Also, eighteen gram positive bacteria, were found to suppress the growth and development of Sclerotina sclerotiorum and Rhizoctonia solani. All of these bacteria isolates grow well in high temperatures (>37 ºC) and twelve grew well at 4 ºC. These bacteria isolates seem to be compatible with T. atroviride. Finally, T. atroviride, a cold tolerant strain, produces a coordinated biochemical response in the presence of different plant pathogens. Our studies indicated that T. atroviride produces cell-wall degrading enzymes at both 22 and 7 C.<br /> <br /> Arizona (Univ of Arizona - Pierson)<br /> Characterization of phenazine regulation and functions in P. aureofaciens 30-84 continues to provide insights into rhizobacterial environmental and community sensing. Molecular and ecological approaches are being used to determine the roles of phenazine antibiotics on biofilm development, root colonization, cell signaling, and pathogen inhibition.<br /> <br /> California (UCR-Becker)<br /> Our research collaboration has developed and optimized research strategies that are applicable to a number of different projects, all dealing with biologically-caused suppression of growth/development of other organisms. With our focus on the identification of the causal agents of nematode population suppression, Prunus replant, and Rhizoctonia damping-off suppression, we attempt to provide the basis for addressing important challenges in agriculture.<br /> <br /> California (UCR-Borneman)<br /> We have identified fungi whose population densities were negatively associated with rootknot nematode population densities. Additionally, we created a series of soils with various levels of peach replant suppression. We are now in the process of performing rRNA gene-based studies to identify the microorganisms involved in these phenomena.<br /> <br /> California (UCR-Stangellini)<br /> Larval stages of shore flies and fungus gnats were shown to ingest and excrete viable oospores of Pythium aphanidermatum, hyphal swellings (sporangia) of P. ultimum, P. splendens, and P. sylvaticum and chlamydospores of Phytophthora ramorum but not sporangia of P. parasitica, P. ramorum or P. capsici. With the exception of oospores of Pythium aphanidermatum, adult shore flies were able to ingest but not excrete viable propagules of any of the oomycetes assayed. Adult fungus gnats did not ingest propagules of any oomycete. Land snails were also demonstrated to ingest and excrete viable chlamydospores, but not sporangia, of P. ramorum.<br /> <br /> California (UCD-Subbarao)<br /> Evaluation of the effects of brassica cover crops on soilborne diseases of strawberries and efficacy of Coniothyrium minitans (commercial product Contans) on lettuce drop caused by Sclerotinia minor are being tested in large experimental plots in California. Preliminary results suggest both to be sustainable non-chemical approaches for soilborne disease management.<br /> <br /> Montana (Montana State Univ. - Jacobsen)<br /> Muscodor albus mycofumigation or use of biorational synthetic M. albus gases has shown potential to control nematodes and a wide range of soil fungal genera including Rhizoctonia, Pythium. Streptomyces, Colletotrichum, and Verticillium. Practical control of several potato diseases caused by these soil microbes has be demonstrated in the field.<br /> <br /> Nebraska (Univ. of Nebraska - Yuen)<br /> Lysobacter enzymogenes C3, a biocontrol agent of plant pathogenic fungi, was found to reduce numbers of the sugarbeet cyst nematode feeding on sugarbeet roots and to promote the growth of sugarbeet plants. This discovery could potentially lead to an environmentally friendly method for managing plant parasitic nematodes. An antibiotic complex produced by C3 was found to be toxic to nematodes. This information could be important in developing and improving C3 as a biocontrol agent. Collaborative research among members of W1147 could eventually lead to better understanding of native population distributions of Lysobacter and how they might be enhanced for disease management.<br /> <br /> New York (Cornell Univ, NYSAES - Abawi)<br /> In 2007, over 1000 soil samples collected from commercial fields, research and demonstration sites were processed for soil health status http://soilhealth.cals.cornell.edu One focus was on relating the collected soil health data, especially root health measurement, to yield and other productivity factors. For example, plots of the vegetable production systems (Conventional, Organic, IPM  Present, and IPM  Future) of the Cornell/NYS IPM Program that have been ongoing since 1995 were planted to snap beans in 2007. Although the data is not statistically significant, the yield of beans grown in the IPM  Future plots had the highest total and marketable bean yield. Previous results of the soil bioassay with beans had also shown that root health is highest (root rot severity is lowest) on roots of plants grown in plots of the Future  IPM system.<br /> <br /> New York (Cornell Univ, NYSAES - Smart)<br /> We are studying the effect of multiple plant activators on field-grown tomatoes to determine if induced systemic resistance (ISR)-inducing biocontrol agents are compatible with other systemic acquired resistance (SAR)-inducing compounds. Utilizing both SAR and ISR-inducing compounds, we have found that SAR-inducing compounds are more effective at controlling foliar diseases of tomato. 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. Additionally, a DNA-based macroarray was developed which can detect over 40 fungal and oomycete pathogens simultaneously. <br /> <br /> Ohio (The Ohio State Univ  McSpadden-Gardener)<br /> By increasing our understanding of the influence of farm management and soil chemistry on biocontrol populations as well as pathogens, we hope to quantify the relative advantages and disadvantages of different practices in terms of their effects on natural biocontrol potential of soils. Identification of novel disease suppressing microorganisms will provide new options for the development of biopesticides to replace methylbromide and other restricted use chemicals with greater toxicity to non-target organisms.<br /> <br /> Washington (USDA-ARS, Pullman - Paulitz)<br /> Phenazine, an antifungal compound produced by Pseudomonas spp., may play an important, but unrecognized role in Rhizoctonia suppression in PNW cereal cropping systems. The biogeography of Pythium and Rhizoctonia in cereal systems is being better understood, especially the influence of soil type, rainfall zone, and cropping system. Conservation fallow may provide a cultural management method for Rhizoctonia in low rainfall zones. Timing of greenbridge sprayout in the spring can reduce damage by Rhizoctonia.<br />

Publications

Impact Statements

1. Our research results on cold tolerant Trichoderma atroviride elucidated the mechanism of hyperparasitism of biological control at room and cold temperatures. This biological control agent offered a safe and environmentally benign means of Armillaria root rot control and an alternative to methyl bromide fumigation.
2. Identification of regulatory networks will allow manipulation of patterns of gene expression to increase pathogen inhibition.
3. Understanding multiple roles of phenazine secondary metabolites in biofilm development will allow improved root colonization and persistence under field conditions.
4. With our focus on the identification of the causal agents of nematode population suppression, Prunus replant, and Rhizoctonia damping-off suppression, we attempt to provide the basis for addressing important challenges in agriculture.
5. These studies are to identify microorganisms involved in Meloidogyne suppressiveness as well as peach and walnut replant suppression, which should lead to the development of new and more sustainable strategies to manage these pests/diseases.
6. Rotations with certain crucifer have previously been proven efficacious for soilborne disease management in conventional strawberry production systems. The current work is demonstrating their efficacy in organic production systems. This work will also provide data on the competitive fitness of Coniothyrium minitans to control S. minor, and may improve its efficacy for lettuce drop control. As there is only one efficacious fungicide for the control of this pathogen, the project will potentially offer additional options for its management.
7. Mycofumigation with Muscodor albus provides a new biologicallybased tool for control of soilborne pathogens for several crops and a wide range of pathogens.
8. Lysobacter enzymogenes C3 could potentially become an environmentally safe tool to integrate with host resistance and management procedures for managing plant parasitic nematodes.
9. Information on the mechanisms of action of Lysobacter enzymogenes C3 against nematodes will be critical towards enhancing the efficacy of the biocontrol agent.
10. Results obtained will be useful in the development of IPM program for detecting, identifying and managing diseases caused by soilborne pathogens that are sustainable, ecological and compatible with soil health long-term management practices.
11. Novel bacterial biocontrol agents have been identified and are being tested for potential biopesticide development.
12. The relative benefits of different organic transition strategies on soilborne disease suppression and crop productivity can now be considered in a cost-benefit analysis of different paths of transition to certified organic vegetable farming.
13. Multiple species of Pythium and Rhizoctonia can be quantified in soil using PCR technology. This will lead to better diagnostics.
14. Cropping systems (crop rotation and fallow) affect species of Pythium and Rhizoctonia, both abundance and population density.
15. Phenazine producers may play a role in Rhizoctonia suppression in the PNW.
16. Genes for Type III secretion systems have been identified in biocontrol Pseudomonas spp. from PNW, and may play a role in biological control or root colonization.
17. Isolates of the causal agent of take-all vary in their sensitivity to phloroglucinol, an antifungal compound involved in biological control.
18. Fallow (chemical and mechanical) may reduce Rhizoctonia activity in low-rainfall zones.
19. Genes that may play a role in root colonization have been identified, sequenced, and mutants tested in the greenhouse.
20. Carryover inoculum from volunteer and weeds can be reduced by proper herbicide sprayout timing before planting.

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