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 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. Demonstration of the optimal time (2-3 weeks before planting) for the sprayout of the greenbridge to reduce Rhizoctonia in cereal-based production aligns with Components 4 because it helps to minimize the number of herbicide applications. Demonstration that Pythium irregulare is strongly associated with legume crop rotations aligns with Components 1 because it allows growers to determine disease risk from Pythium and the best rotation practices to combat diseases. Demonstration that Fusarium crown rot of wheat caused by F. pseudograminearum and F. culmorum is not affected by the previous crop aligns with Components 4 because it allows growers to make sustainable disease management decisions. Demonstration that DAPG metabolism genes are induced in wheat roots during interactions with DAPG-producing Pseudomonas aligns with Component 3. Results show that wheat roots rapidly detoxify DAPG, and indicate a host pathway that can be modulated to enhance interactions with DAPG-producing bacteria. Demonstration that loss of COI1 activity in tomato was correlated with enhanced susceptibility to P. aphanidermatum aligns with Component 3 because it shows the first link between COI1 and tolerance to necrotrophic root pathogens in a crop plant. Demonstration of Rhizoctonia and Pythium tolerance in the wheat, Scarlet-Rz1 aligns with Component 3 because it is the first known source of genetic resistance to these two pathogens in a commercially-available variety.
1. Optimal time for sprayout of greenbridge is 2-3 weeks before planting. There is no cultivar resistance or chemicals for control of Rhizoctonia root rot, a major disease of cereals. The most effective cultural means of managing this disease is greenbridge control, which involves removing weeds and volunteers with herbicides well before planting in the spring. But when is the optimal time to sprayout the greenbridge? ARS scientists at Pullman, WA conducted field experiments in naturally infested plots with different timings of the herbicide glyphosate. The optimum time for spraying is at 2 to 3 weeks before planting; longer times did not significantly increase yield or reduce disease. This accomplishment aligns with Components 4, Problem Statement 4A and 4C of NP 303.
2. The root pathogen Pythium irregulare is strongly associated with legume crop rotations. Pythium irregulare is one of the most virulent species of Pythium on wheat. ARS scientists at Pullman, WA, using species-specific primers they developed for quantitative PCR, surveyed variety testing and grower sites throughout the wheat growing area. The highest levels of P. irregulare were found in wheat that had been previously cropped with lentils. This pathogen is also virulent on lentils. This information is useful for growers to determine disease risk and crop rotation. This accomplishment aligns with Components 1, Problem Statements 1A and 1B and Component 2, Problem Statement 2C of NP 303.
3. Fusarium crown rot is not affected by the previous rotation crop. Crop rotation is an effective tool to manage some root diseases. Fusarium crown rot, caused by Fusarium pseudograminearum and F. culmorum, is re-emerging as a serious problem even in higher rainfall areas, especially on hard red wheat managed for high protein. ARS scientists at Pullman, WA, performed 3 years of field rotation trials, and assayed disease and levels of the pathogens in the soil using DNA techniques. Common rotation crops, such as barley, canola, and peas, did not influence the disease in the following year on wheat, showing that the Fusarium pathogen survives for at least 1 year in the absence of a host. This is important information for growers to make management decisions on rotation crops. This accomplishment aligns with Components 4, Problem Statement 4A and 4C of NP 303.
4. Genes for the metabolism of the antibiotic 2,4-diacetylphloroglucinol (DAPG) are induced in wheat roots during interactions with DAPG-producing Pseudomonas. Production of 2,4-diacetylphloroglucinol is a major mechanism of biocontrol of root diseases. Molecular changes that occur in wheat during early stages of root colonization by biocontrol bacteria are deemed critical for establishing successful biocontrol interactions, however very little is known about such molecular changes. ARS scientists at Pullman, WA used microarray analysis to determine which wheat genes show altered expression in response to DAPG-producing P. fluorescens strain Q8r1-96, a well-studied biocontrol agent of wheat root diseases. Genes encoding metabolic enzymes were induced within 48 h of inoculation with strain Q8r1-96. The findings suggest that wheat roots rapidly detoxify DAPG, and indicate a host pathway that might be manipulated to enhance interactions with DAPG-producing biocontrol bacteria. This accomplishment aligns with Component 3, Problem Statement 3A of NP 303.
5. Pythium susceptibility is increased in coi tomato mutant. The jasmonate pathway regulator COI1 has been implicated in innate immunity (tolerance) to wounding insects in tomato, but its role in tolerance to necrotrophic soilborne pathogens has not been defined. ARS scientists at Pullman, WA in collaboration with a scientist at Michigan State University tested a tomato mutant lacking a functional COI1 for changes in tolerance to Pythium aphanidermatum. Loss of COI1 activity in tomato was correlated with enhanced susceptibility to P. aphanidermatum. This finding is the first report of a link between COI1 and tolerance to necrotrophic root pathogens in a crop plant, and suggests a mechanism by which tolerance can be enhanced in host roots. This accomplishment aligns with Component 3, Problem Statement 3A of NP 303.
6. Rhizoctonia and Pythium tolerance found in a non-genetically modified wheat, Scarlet-Rz1. All varieties of wheat are susceptible to the soilborne pathogens Rhizoctonia solani, R. oryzae, Pythium ultimum and P. irregulare, and genetic resistance to these pathogens has been elusive until now. ARS scientists at Pullman, WA in collaboration with a scientist at Washington State University tested a wheat genotype called Scarlet-Rz1 generated by chemical mutagenesis for tolerance to the four pathogens. Six generations derived from Scarlet-Rz1 had significantly lower disease severity ratings and less root loss when infected with the pathogens as compared to native Scarlet. This novel, stable source of genetic resistance to hard-to-control soilborne pathogens offers a sustainable means of combating Rhizoctonia and Pythium, and provides a new source of resistance for wheat improvement programs. This accomplishment aligns with Component 3, Problem Statement 3B of NP 303.
7. Identifying the molecular basis of root colonization by biocontrol bacteria: The genetic basis for differences in rhizosphere colonization among closely related 2,4-diacetylphloroglucinol-producing biocontrol strains of Pseudomonas fluorescens is not known. ARS scientists at Pullman, WA in collaboration with a scientist at Washington State University sequenced and analyzed a new P. fluorescens Q8r1-96-specific locus, ssh53. Sequence analysis revealed that the locus is absent from genomes of closely related Pseudomonas spp. and forms part of a large gene cluster for cell surface lipopolysaccharide biosynthesis. The mutant is defective in biofilm formation, exhibits altered colony morphology, and is essential for survival of P. fluorescens Q8r1-96 on wheat roots in non-sterile soil. The results implicate bacterial strain-specific surface interactions with the host as critical to the premier colonization phenotype. This accomplishment aligns with Component 4, Problem Statement 4B of NP 303.
8. The type III secretion locus in P. fluorescens Q8r1-96 is required for root colonization: The Type III secretion apparatus common to pathogens of plants and animals is a critical determinant in the interaction of these bacteria with their hosts. ARS scientists at Pullman, WA sequenced genes encoding components of type III secretion (TTSS) and determined their role in the premier biocontrol strain P. fluorescens Q8r1-96. Q8r1-96 carries a hrp/hrc locus highly similar to its counterpart in the plant pathogen P. syringae pv. phaseolicola, including homologues of all genes of the hrp/hrc cluster, and these genes are required for efficient root colonization. The results demonstrate that molecular interactions not only with the pathogen, but also with the plant, are involved in this premier biocontrol interaction. This accomplishment aligns with Component 4, Problem Statement 4B of NP 303.
9. Construction of new genetically engineered microbial agents for biocontrol of plant fungal diseases: Antibiotics produced by biocontrol agents typically function most effectively against a limited range of fungal pathogens. To expand the target range of the biocontrol strain Pseudomonas. fluorescens Q8r1-06, ARS scientists at Pullman, WA in collaboration with a scientist at Washington State University constructed transgenic derivatives carrying the pyrrolnitrin biosynthetic operon and a gene encoding 1-aminocyclopropane-1-carboxylate (ACC)-deaminase, which confers plant growth-promoting activity. The pyrrolnitrin-producing derivative gained the ability to inhibit Rhizoctonia solani AG-8 and Fusarium pseudograminearum. The enhanced strains are expected to have improved biocontrol and plant growth-promoting activity under greenhouse and field conditions. This accomplishment aligns with Component 4, Problem Statement 4A of NP 303.
10. Identification of root-lesion nematodes: Published Pratylenchus species-specific primers are limited in ability to clearly differentiate between P. neglectus and P. thornei especially in mixed cultures derived from extractions of soil naturally infested with different plant-parasitic and saprophytic species. ARS collaborators at Oregon State University, Pendelton, OR in collaboration with ARS scientists at Pullman, WA and Beltsville, MD optimized new species-specific primers for detecting and providing ratios of prevalence for the two Pratylenchus species, which are important to dryland agriculture in the Pacific Northwest. A multiplexing assay was also developed to enable each species to be quantified as well as identified from a single soil extract using real-time PCR. Results are being calibrated against results from commercial nematode diagnostic laboratories, with the intent that this technology will be transferred into commercial labs equipped for PCR procedures but which do not provide morphometric-based species-level identifications of Pratylenchus. This accomplishment aligns with Component 1, Problem Statement 1B of NP 303.
11. Root diseases in conservation farming systems: Changing patterns for disease occurrence and severity are monitored during the transition from traditional winter wheat-summer fallow rotations to conservation-oriented systems including chemical fallow, annual spring cropping, and three year rotations in low-precipitation environments. ARS collaborators at Oregon State University, Pendelton, OR evaluated diseases in two long-term cropping system transition studies. They showed that Rhizoctonia root rot, Fusarium crown rot and root-lesion nematodes were the most damaging diseases in studies near Moro and Heppner, Oregon. The most important finding was that populations of root-lesion nematodes were much lower following production of barley compared to wheat, canola, mustard or winter pea. During early spring, the root lesion nematodes in chemical and cultivated summer fallow were detected in high populations at soil profile depths greater than typically used to survey nematodes in field crops. At Moro, in an experiment evaluating eight replicated crop management systems in a low precipitation (12 inch) region, yield of winter wheat over a 3-year period was highly and inversely correlated with populations of Pratylenchus neglectus. This information corresponds to experiences of farmers who adopted conservation practices but have been disappointed by lower wheat yields following Brassica crops or summer fallow compared to wheat following barley. This accomplishment aligns with Component 4, Problem Statement 4A of NP 303.
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 Community 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 four 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 BCC instructors; iii) development of and participation in on-reservation summer science camps; and iv) employing Native American, Latino and rural high school summer science interns in ARS laboratories. 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 2007 to May, 200; 3) sponsorship of and participation in the Second Annual Skwant Life Science Camp at the Pascal Sherman Indian School (Skwant is translated as ‘Waterfalls’), June 2007 (two 1-week sessions for 5th-6th and 7th-8th graders and a total of 70 campers); and 4) molecular biology training for 6 summer high school interns (July 2008) in ARS labs at Pullman. This award-winning program is promoting economic development on the Colville Reservation, enhancing science awareness among Native American, Latino and rural youth, and training the next generation of scientists. Native American reservations and rural communities throughout the Pacific Northwest are requesting expansion of this ARS program to their communities.