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United States Department of Agriculture

Agricultural Research Service



2011 Annual Report

1a. Objectives (from AD-416)
The long-term objective of this program is to develop biologically based technology for controlling soilborne pathogens of wheat, barley, and Brassicas, grown as part of cereal-based production systems. Five specific objectives will be addressed over the next five years. Objective 1: Evaluate the genotypic diversity between species and within pathogen populations of root pathogens in direct-seeded cereal-based cropping systems. Objective 2: Identify and characterize microorganisms and mechanisms active in the suppression of soilborne pathogens by rhizobacteria. Objective 3: Identify the determinants responsible for differential rhizosphere competence among genotypes of DAPG-producing Pseudomonas fluorescens strains. Objective 4: Identify and characterize the molecular mechanisms of host-microbe interactions, including the action of host genes governing disease resistance and biological control. Objective 5: Identify and develop biocontrol technology for suppression of root pathogens in cereal-based cropping systems.

1b. Approach (from AD-416)
Biological control of root pathogens Gaeumannomyces, Pythium, Rhizoctonia, and Fusarium by naturally occurring and genetically engineered microorganisms will be developed and quantified in different soils. The genetic determinants and molecular mechanisms responsible for root colonization and pathogen suppression will be characterized with emphasis on the genetics and regulation of phenazine and phloroglucinol biosynthesis. Structural loci for these anti-fungal metabolites will be used to engineer improved biocontrol agents. The genetic and physiological diversity of populations of the root pathogens, and influence of cropping system on pathogen populations and diversity will be determined. New sources & mechanisms of resistance will be identified. Practical root diseases control will be accomplished by both maximizing the activity of natural biocontrol agents. Formerly 5348-22000-012-00D (2/07).

3. Progress Report
Most wheat, barley and biofuel crops are infected by soilborne fungal pathogens and parasitic nematodes that reduce yields 10-30% annually. Diseased crops cannot take full advantage of fertilizers and irrigation water, and unused nitrates move into surface and ground water. The goal of this project is to develop biologically-based technology for controlling root diseases of wheat, barley and biofuel brassica crops. Progress was made on all five objectives and their subobjectives, all of which fall under National Program 303 and encompass Component 1 Problem B, Component 2 Problem C, Component 3 Problem B, or Component 4 Problem A and B. Under Objectives 1.C and 4.C, we made significant progress in identifying Pacific Northwest wheat varieties with aluminum tolerance. This research aligns with Components 1 and 3 because it describes a new problem of wheat in the Pacific Northwest and identifies varieties that can be grown in low pH, high aluminum PNW soils in order to solve the problem. Under Objectives 1.A, B and C, we made significant progress in conducting a disease survey that showed major infestations of cereal cyst nematode (CCN) in eastern Washington State wheat. This research aligns with Components 1, 2 and 3 because it identifies a new disease threat to Pacific Northwest wheat, provides new fundamental information about the ecology and epidemiology of CCN, and identifies genetic approaches to deal with the disease threat. Under Objectives 1.C, ARS scientists in Pullman, WA made significant progress in demonstrating how herbicides used with Clearfield wheat predisposes future barley crops to Rhizoctonia root rot. This research aligns with Component 1 and 4 because it shows how management practices can impact the severity of Rhizoctonia root rot of wheat and highlights how the use of new technology can exacerbate an already serious disease problem. Under Objectives 1.A, we made significant progress in developing a molecular diagnostic assay for the root lesion nematodes Pratylenchus thornei, which along with P. neglectus accounts for an estimated $51 million annual loses of wheat in the Pacific Northwest. This research aligns with Component 1 because it allows rapid detection and quantification of this nematode and an assessment of risk prior to planting. Under Objectives 4.C, we made significant progress in demonstrating that the heritability of Rhizoctonia tolerance in six new wheat genotypes is a dominant trait in all genotypes. This research aligns with Component 3 because this resistance provides a novel approach to suppress Rhizoctonia root rot in wheat.

4. Accomplishments
1. Identification of aluminum tolerant wheats. Low pH and aluminum toxicity cause root toxicity and significant yield loss in blue-grass/wheat rotations in the Pacific Northwest (PNW). In 2008, growers reported severely stunted wheat in Spokane and Latah counties, covering about 30,000 acres. ARS scientists at Pullman, WA identified the problem as low pH and aluminum toxicity resulting from 60 years of high ammonium inputs. Use of large amounts of lime to alleviate the problem is not economically viable on a low value crop. Screening for aluminum tolerant wheat varieties in the field identified a number of PNW winter varieties such as Madsen and spring varieties such as JD and Babe, as a solution to this problem.

2. Cereal cyst nematode is a greater threat than previously thought. Cereal cyst nematode (CCN) is the most important nematode pathogen of wheat worldwide. It has been a problem in NE Oregon, but the extent of infestation in eastern Washington was unknown. ARS scientists at Pullman, WA conducted a survey in the summer of 2010 and found extensive infestations of CCN with classic symptoms throughout the high precipitation annual cropping area of eastern Washington. This study indicates that CCN poses a greater threat than previously thought. Developing varieties with genetic resistance (Cre genes) is a viable solution to this problem.

3. Certain herbicides predispose barley to Rhizcoctonia damage. Over the last 5 years, Clearfield wheat with resistance to Group 2 imidazolinone amino acid synthesis (ALS) inhibitor herbicides has been widely planted in the Pacific Northwest. However, barley is sensitive to residual levels of these herbicides in the soil, which has lead to plant-back restrictions and severely reduced the acreage of barley. ARS scientists at Pullman, WA in collaboration with researchers at Washington State University showed that barley plants exposed to sublethal levels of Beyond and Olympus herbicides were predispose to Rhizcoctonia damage. These findings explain for the first time why barley does poorly after wheat treated with these herbicides, even after the plant-back period.

4. Plant growth-promoting rhizobacteria (PGPR)-induced wheat root defense gene expression varies with cultivar. Wheat cultivar-dependent root responses to Pseudomonas fluorescens might be a factor in the variability of plants to PGPR biocontrol bacteria. ARS scientists at Pullman, WA monitored the expression of fifteen defense gene homologues in roots of cultivars Tara and Buchanan after colonization with wild-type strain Q8r1-96, strain 4C5 (phloroglucingol-null mutant of Q8r1-96) or strain PST (type III secretion system deletion mutant) using molecular diagnostic assays. There were few differences in gene induction among the strains, but roots of cultivar Buchanan displayed an earlier and more comprehensive pattern of gene induction than did Tara. These findings demonstrate that host gene expression underlying biocontrol is dependent on the host cultivar. As a result, biocontrol maybe improved by selecting cultivars that respond rapidly to PGPR.

5. Rhizoctonia soil populations that pose planting risks. Rhizoctonia solani AG-8 is one of several major soilborne pathogens in cereal production systems in the Pacific Northwest, and the relationship between plant health and soil populations of this pathogen need to be defined for grower risk advisories. ARS scientists at Pullman, WA quantified the growth and health of barley after pathogen challenge in the greenhouse. Inhibition of shoot and root growth, and increased root damage were noted at 20 propagules per gram (ppg), and injury increased with 50-200 ppg of pathogen. In the absence of a feasible design for field trials, this research has defined planting risk threshold populations for Rhizoctonia in barley fields.

6. New Pseudomonas strains suppressive to necrotrophic soilborne fungal pathogens of wheat. The limitations of tillage and herbicide application for greenbridge control needed to manage root rot pathogens of wheat in the Pacific Northwest, include fuel inputs, soil erosion and timing of applications between plantings. ARS scientists at Pullman, WA have identified nine new strains of Pseudomonas spp. that reduced damage caused by necrotophic soilborne fungal pathogens in greenhouse assays. Six strains suppressed disease symptoms of Rhizoctonia solani AG-8, two strains suppressed damage by Pythium ultimum and one strain was suppressive to both pathogens. The strains are being considered for alternative biotic control of soilborne fungal pathogens of wheat.

7. Rhizoctonia tolerance in wheat. Development of genetic resistance is a sustainable approach to curtailing losses caused by soilborne fungal pathogens in cereal production systems of the Pacific Northwest (PNW). ARS scientists at Pullman, WA characterized the heritability of Rhizoctonia tolerance in six new wheat genotypes using greenhouse assays. Tolerance was inherited as a dominant trait in all genotypes. The genetic resources generated in this project are leading to an understanding of wheat’s susceptibility to Rhizoctonia. This research will result in PNW-adapted, Rhizoctonia-tolerant wheat cultivars that yield better than current cultivars in Rhizoctonia infested fields.

8. Molecular diagnostic assay for Pratylenchus thornei. The root lesion nematodes Pratylenchus thornei and P. neglectus affect up to 60% of dryland wheat fields and account for an estimated $51 million annual loses of wheat in the Pacific Northwest. ARS scientists at Pullman, WA in collaboration with researchers at Oregon State University developed a rapid and specific molecular assay for P. thornei. The assay was used to quantify P. thornei populations in at-risk field soils, and results correlated well to data obtained using conventional counting methods. This assay now permits comprehensive soil surveys, risk assessment in a field and rapid screens for Pratylenchus-resistant wheat.

9. Diversity and evolution of the phenazine biosynthesis pathway. Phenazines are versatile secondary metabolites that function as antibiotics in the biocontrol of plant pathogens. ARS scientists at Pullman, WA in collaboration with researchers at Washington State University determined the distribution and evolution of phenazine genes in representative bacterial species that produce these compounds. Most phenazine producers are soil-dwelling and/or plant-associated species. Genetic analyses of sequences of the key phenazine biosynthesis (phzF) and housekeeping genes revealed that the evolution and dispersal of phenazine genes occur by mechanisms ranging from conservation in some species to horizontal gene transfer in others. DNA extracted from soil surrounding the roots of cereal crops and screened for the presence of phzF contained sequences consistent with the presence of a diverse population of phenazine producers in commercial farm fields located in central Washington State, providing the first evidence of USA soils enriched in indigenous phenazine-producing bacteria.

10. Structural and functional analysis of the type III secretion system (T3SS) of the biocontrol bacterium Pseudomonas fluorescens Q8r1-96. P. fluorescens Q8r1-96 represents a group of root-inhabiting bacteria responsible for the natural biocontrol of take-all disease of wheat. ARS scientists at Pullman, WA in collaboration with researchers at Washington State University analyzed the genome of Q8r1-96 to identify genes that might account for the ability of this strain to aggressively colonize wheat roots. In Q8r1-96 and in 29 of 30 related strains, we identified a gene cluster encoding a T3SS, which are structures that enable bacteria to introduce effector proteins into the cells of their hosts, modifying host cell behavior. The Q8r1-96 genome encodes three effectors that were secreted in culture and injected into plant cells. The genes were expressed by bacteria on roots, but mutants lacking a functional T3SS were not altered in their colonization ability. These studies are providing fundamental new insight into how biocontrol bacteria interact with their host.

Review Publications
De Luna, L.Z., Kennedy, A.C., Hansen, J.C., Paulitz, T.C., Gallagher, R.S., Fuerst, E.P. 2011. Mycobiota on wild oat (Avena fatua L.) seed and their caryopsis decay potential. Plant Health Progress. doi:10.1094/PHP-2011-0210-01-RS.

Kwak, Y., Han, S., Thomashow, L.S., Topham, J., Paulitz, T.C., Kim, D., Weller, D.M. 2011. Saccharomyces cerevisiae genome-wide mutant screen for sensitivity to 2,4-diacetylphloroglucinol, a biocontrol antibiotic produced by Pseudomonas fluorescens. Applied and Environmental Microbiology. 77(5):1770-1776.

D'Aes, J., Khuong, H.G., De Maeyer, K., Pannecoucque, J., Forrez, I., Ongena, M., Dietrich, L.E., Thomashow, L.S., Mavrodi, D.V., Mavrodi, O.V., Hofte, M. 2011. Biological control of Rhizoctonia root rot on bean by phenazine- and cyclic lipopeptide-producing Pseudomonas CMR12a. Phytopathology. 101(8):996-1004.

Mavrodi, D.V., Joe, A., Mavrodi, O., Hassan, K.A., Weller, D.M., Paulsen, I.T., Loper, J.E., Alfano, J.R., Thomashow, L.S. 2011. Structural and functional analysis of the type III secretion system from Pseudomonas fluorescens Q8r1-96. Journal of Bacteriology. 193(1):177-189.

Okubara, P.A., Jones, S.S. 2011. Seedling tolerance to Rhizoctonia and Pythium in wheat chromosome group 4 addition lines from Thinopyrum spp. Canadian Journal of Plant Pathology. 33(3): 415-422.

Li, Q., Mavrodi, D.V., Thomashow, L.S., Roessle, M., Blankenfeldt, W. 2011. Ligand binding induces an ammonia channel in 2-amino-2-desoxyisochorismate (ADIC) synthase PhzE. Journal of Biological Chemistry. 286(20):18213-18221.

Paulitz, T.C. 2011. Disease management. In: Hatfield, J.L., Sauer, T.J., editors. Soil management: building a stable base for agriculture. Madison WI: American Society of Agronomy and Soil Science of America. p. 265-274.

Yin, C., Jones, K.L., Peterson, D.E., Garrett, K.A., Hulbert, S.H., Paulitz, T.C. 2010. Members of soil bacterial communities sensitive to tillage and crop rotation. Soil Biology and Biochemistry. 42(12):2111-2118.

Chekali, S., Gargouri, S., Paulitz, T.C., Nicol, J., Rezgui, M. 2011. Effects of Fusarium culmorum and water stress on durum wheat in Tunisia. Crop Protection. 30(6):718-725.

Paulitz, T.C., Steffenson, B.J. 2011. Biotic stress in barley: disease problems and solutions. In: Ullrich, S.E., editor. Barley: improvement, production, and uses. Ames, IA: Wiley-Blackwell. p. 307-354.

Last Modified: 2/23/2016
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