2010 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).
Most wheat and barley is infected by root diseases including take-all, Rhizoctonia and Pythium root rots, common root rot, and Fusarium foot rot caused by soilborne fungi, which 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 pathogens of crops, especially wheat and barley, grown as part of cereal-based production systems in the Pacific Northwest. Brassica crops, such as canola, are grown as rotation crops in dryland areas, but have the potential to significantly expand in acreage because of increased demand for biodiesel, edible oils, and seedmeal. Brassicas are attacked by many of the same fungi that infect cereals. Project objectives include:.
1)evaluate the genotypic diversity between species and within populations of root pathogens in direct-seeded cereal-based cropping systems;.
2)identify and characterize microorganisms and mechanisms active in the suppression of root pathogens by rhizobacteria;.
3)identify the determinants responsible for differential rhizosphere competence among genotypes of 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas fluorescens strains;.
4)identify and characterize the molecular mechanisms of host-microbe interactions, including the action of host genes governing disease resistance and biological control; and.
5)identify and develop biocontrol technology for suppression of root pathogens in cereal-based cropping systems. This project encompasses all components of NP 303. Characterization of bacterial communities in the soil by cultureless techniques such as pyrosequencing aligns with Components 4 because it identifies new components of communities that can play a role in soil health and suppression of root diseases. Identification of minor gene resistance in wheat against Fusarium crown rot aligns with Component 3 because it speeds the deployment of resistance found in Australian and Middle Eastern lines into wheat varieties adapted to the Pacific Northwest. Development and application of a molecular diagnostic assay for the pathogen causing Fusarium wilt of spinach aligns with Component 1 because it identifies whether a field harbors a problematic level of the pathogen before sowing spinach. The survey of Rhizoctonia species from wheat throughout the U.S. aligns with Component 2 because it will contribute to our understanding of the geographical distribution and biology of these important pathogens and will aid in developing proper disease management strategies. Development of Real-time PCR assays to quantify Rhizoctonia and Pythium aligns with Component 1 because it allows for DNA to be quantified from soil samples collected in the field and a relative disease risk for root rot to be assigned to a field.
Canola attacked by Rhizoctonia stem rot. Canola is grown in rotation with wheat in the Pacific Northwest, and has potential as both a biofuel and oilseed crop. ARS researchers at Pullman, WA discovered a group of the pathogenic fungus Rhizoctonia (R. solani AG 2-1) that causes post-emergence death of seedlings and wirestem symptoms. This same pathogen is also pathogenic to other broadleaf rotation crops. In collaboration with WSU scientists, they are screening Brassica germplasm for resistance to this disease and surveying the region using molecular detection and quantification methods. This disease poses a great threat to the expansion of sustainable canola production needed to meet the biofuel needs of the Pacific Northwest.
Low pH and aluminum toxicity cause root toxicity and significant yield loss in blue-grass wheat rotations in the Pacific Northwest. 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 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. ARS scientists are screening aluminum tolerant wheat varieties and germplasm as a solution to this problem.
Minor gene QTL with resistance to Fusarium crown rot found in mapping population. Fusarium crown rot is an important disease in dryland low-rainfall production areas of the Pacific Northwest, causing an average of 9.5% yield loss. ARS scientists at Pullman, WA identified significant QTLs or minor gene resistance for this disease from a mapping populations screened in the greenhouse and field, using methods they developed. The QTL was localized to Chromosome 3B. Using Marker Assisted Selection, this QTL can be moved into locally adapted varieties.
Biocontrol agents-induced wheat root defense gene expression varies with cultivar. Defense genes are induced in roots of wheat cultivar Finley by biocontrol bacteria, but it is not known how conserved this response to biocontrol bacteria is in other cultivars. ARS scientists at Pullman, WA monitored the expression of 18 defense genes in roots of cultivars Tara and Buchanan after colonization with the biocontrol strain Q8r1-96 using molecular diagnostic assays. The two cultivars differed in their patterns of gene induction, and differed from Finley in their response to the biocontrol bacteria. The findings indicate that gene expression underlying biological control is dependent on the host cultivar, with implications for field applications.
The jasmonic acid signal pathway is linked to innate immunity against Pythium in tomato. Many crop plants are susceptible to the soilborne pathogens that cause root rot, and although genetic resistance to these pathogens will benefit growers in the Pacific Northwest and throughout the world, root-dependent resistance pathways are poorly defined at the molecular level. ARS scientists at Pullman, WA in collaboration with researchers at Michigan State University used molecular diagnostic assays to quantify Pythium in roots of tomato plants with or without a functional jasmonic acid signal pathway. Soils and roots from plants lacking a functional pathway harbored about ten-fold more pathogen than those of the wild type, and also supported a second Pythium pathogen. The findings indicate that the jasmonic acid signal pathway has a role in Pythium resistance, and this genetic resistance also reduces pathogen buildup in soils.
Quantification of Fusarium wilt pathogen of spinach in Washington soils. The pathogen causing Fusarium wilt of spinach is exceptionally persistent in the soils, and Pacific Northwest spinach growers need to know whether their fields harbor problematic levels of the pathogen before they plant. ARS scientists at Pullman, WA in collaboration with researchers at Washington State University used a rapid and sensitive diagnostic assay for monitoring pathogen populations in soils before and after soil amendments. Pathogen populations correlated with disease severity in non-amended soils, but were not substantially reduced when amendments were used, even though disease severity was decreased. The diagnostic assay is an important tool to predict pre-planting risk but is not useful for evaluating soil amendments as a control strategy.
Rhizoctonia soil populations that pose planting risks. Rhizoctonia root rot is one of several major soilborne pathogens in wheat production systems in the Pacific Northwest, USA, 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 wheat after challenge with Rhizoctonia in the greenhouse. Inhibition of shoot and root growth, and increased root damage were noted at 50 propagules per gram (ppg) of pathogen for a susceptible cultivar and at 100-200 ppg for a Rhizoctonia-resistant cultivar. In the absence of a feasible design for field trials, this research has defined planting risk threshold populations for Rhizoctonia in field soils.
Survey of Rhizoctonia from wheat fields nationwide. Rhizoctonia root rot of wheat is caused by a complex of Rhizoctonia species, each having different degrees of virulence on cereals and varying host ranges. The species involved in the Rhizoctonia root rot complex in Washington State have been well documented; however, information is lacking as to the distribution of this pathogen in other wheat production areas in the U.S. ARS scientists at Pullman, WA in collaboration with researchers at a crop protection company collected Rhizoctonia isolates from wheat fields throughout the country and identified them using sequence analysis of the ITS region of the rDNA. In the first phase of the survey, 73 soil samples were processed and nearly 40 isolates were identified by sequence analysis of the ITS gene, with R. oryzae and R. solani AG-2-1 being the most common. R. oryzae has been implicated as a major component of the Rhizoctonia root rot complex, while R. solani AG-2-1 is a serious pathogen of brassica and legume crops. Information about the diversity of Rhizoctonia species will contribute to our understanding of the geographical distribution and biology of these pathogens and will aid in developing proper disease management strategies.
Quantification of Pathogens in Soil. Real-time PCR assays have been developed to quantify Rhizoctonia and Pythium in soil and plants. However, data is needed to correlate DNA quantities with disease risk. ARS Scientists in Pullman, WA established a greenhouse studies to evaluate the disease impact of varying levels of Pythium inoculum and to correlate this information with DNA quantities of the pathogen in the soil. Varying quantities of DNA were found in the soil; however, this did not correspond to a measurable difference in the amount of disease on the plants. Further investigations will be required to obtain the proper disease response from these pathogens. When completed, this information will allow for DNA to be quantified from soil samples collected in the field and a relative disease risk for Pythium root rot to be assigned. Since a diagnostic lab in Idaho has begun using our technology for detection of these pathogens, developing a risk model would greatly supplement this tool.
Diseased roots select for bacteria antagonistic to pathogens. Rhizoctonia root rot on wheat can be suppressed by natural microbes present in the soil. A pinch of soil contains thousands of species, but only a few can be cultured and identified. Using a new technique called pyrosequencing, ARS scientists at Pullman, WA in collaboration with researchers at Washington State University found species of Flavobacterium and Enterobacteraceae that significantly increased on diseased roots compared to healthy roots. These bacteria may play a role in improving soil health and suppression of root diseases.
5.Significant Activities that Support Special Target Populations
ARS scientists at Pullman, WA lead a science outreach and engagement program, with participation by USDA, NRCS, Washington State University Cooperative Extension, WSU Plant Pathology Department, and Bellevue College, to The Confederated Tribes of the Colville Reservation and the region surrounding Omak, WA. This is one of the most remote, poorest and underserved regions in Washington State. The program entitled “Pumping-Up the Math and Science Pipeline: Grade School to College” has five components: i) development of energy independence on the Colville Reservation through the production of biofuels; ii) hands-on science education in reservation schools by ARS research scientists, WSU faculty and BC instructors; iii) development of and participation in on-reservation summer science camps; iv) supporting Native American, Hispanic, African American and rural high school summer science interns in ARS laboratories, and v) mentoring WSU undergraduates participating in the College Assistance Migrant Program (CAMP). Examples of these activities include:.
1)field tests of spring and winter canola varieties and biodiesel production on Colville Reservation land;.
2)monthly visits to Nespelem Elementary School and Pascal Sherman Indian School on the Colville Reservation to present science modules, October 2009 to May, 2010;.
3)sponsorship of and participation in the Third Annual Skwant Life Science Camp for 5th-8th graders at the Pascal Sherman Indian School (Skwant is translated as ‘Waterfalls’), June 21-25, 2010, (total of 40 campers); and.
4)molecular biology training for 7 summer high school interns and one high school teacher (July 2010) in ARS labs at Pullman. The award-winning Pipeline Program is promoting economic development on the Colville Reservation, enhancing science awareness among Native American, Hispanic and rural youth, and training the next generation of STEM professionals. Native American reservations and rural communities throughout the Pacific Northwest have requested expansion of this ARS program to their communities.