Project : USDA ARS

ARS Home » Northeast Area » Frederick, Maryland » Foreign Disease-Weed Science Research » Research » Research Project #440440

Research Project: Integrative Genomic and Biological Approaches to Detect and Manage Emerging Foreign Fungal and Oomycete Plant Pathogens

Location: Foreign Disease-Weed Science Research

2024 Annual Report

Objectives
Objective 1: Generate and utilize genomic and proteomic sequence information of emerging and threatening fungal plant pathogens to develop diagnostic assays. Sub-objective 1.A: Develop accurate and rapid means for the identification and detection of foreign fungal plant pathogens. Subobjective 1.B: Determine origins and distribution routes of foreign and recently introduced plant pathogens. Sub-objective 2.B: Evaluate chemical control methods for emerging fungal plant pathogens. Objective 2: Identify factors involved in fungal pathogen infection and virulence. Sub-objective 2.A: Determine environmental factors affecting sporulation of fungal plant pathogens. Sub-objective 2.B: Evaluate chemical control methods for emerging fungal plant pathogens. Sub-objective 2.C: Identify genes and proteins contributing to virulence in fungal plant pathogens. Objective 3: Screen germplasm and identify resistance genes to emerging and threatening fungal plant pathogens. Sub-objective 3.A: Screen germplasm for resistance to foreign fungal plant pathogens. Sub-objective 3.B: Identify accession-specific resistance genes for emerging and threatening fungal plant pathogens. Sub-objective 3.C: Identify new races of fungal pathogens that can be controlled by existing resistance genes.

Approach
Genomic sequence information will be generated from host plants and foreign fungal plant pathogens. Bioinformatic analyses will be conducted to identify resistance genes, pathogenicity factors, markers for evaluating pathogen populations, and unique targets for DNA-based diagnostic assay development. Pathogen proteins or isoforms will be identified and used to generate antibodies to develop immunodiagnostic assays. Fungicides will be evaluated for their efficacy in controlling emerging fungal plant pathogens. Controlled environment growth chambers will be used to determine the effect of temperature and humidity on the sporulation of fungal plant pathogens. Containment greenhouses will be utilized to screen plant germplasm with foreign fungal plant pathogens to identify sources of host resistance.

Progress Report
Under Objective 1, Sub-objective 1.A, custom-designed antibodies for the development of immunoassays to detect the boxwood blight fungal pathogens Calonectria pseudonaviculata and C. henricotiae were received from a commercial vendor. Antibodies are currently being tested against boxwood leaves infected with different isolates of boxwood blight pathogens to ensure specificity. Isothermal assay reagents were designed and tested on analytical DNA extract samples on a high throuhput fluorescent cycler. Sensitivity tests were completed satisfactorily and specificity testing with Calonectria isolates are ongoing. Under Sub-objective 1.B: SSR genotypes for 120 isolates of C. pseudonaviculata were generated and analyzed, and phenotypic analysis was initiated. Under Objective 2, Sub-objective 2.A, experiments to compare the effects of relative humidity on sporulation of boxwood blight on different boxwood cultivars are almost completed. Preliminary results indicate that a relative humidity of 80% or above is required for sporulation to occur. This information will be used to predict boxwood blight severity in different regions of the U.S. Additionally, photographs of boxwood blight symptoms were taken for different cultivars and at different stages of infection for use in outreach documents and future publications. Under Sub-objective C.2, we began testing different methods to inoculate peanuts with the causal agent of peanut smut, Thecaphora frezzii. We successfully infected peanuts and are now refining our methods to increase the incidence of infection. We also assembled a reference quality genome for T. frezzii and obtained RNA from the mycelium of multiple isolates that is guiding the annotation. We determined that exposure to light plays a critical role in the production of a phytotoxin produced by C. glycines. Therefore, we grew eight C. glycines isolates under light and dark regimes and used the resulting tissue for RNA sequencing and proteomic analyses. Approximately 1,500 genes were significantly differentially expressed in the transcriptomics analysis, while 2,000 proteins had significantly different abundances under the different lighting regimes. At least one secondary metabolite gene cluster has been identified as a putative virulence factor for C. glycines, but the genes in it are only expressed in lit conditions. Transcriptomes were generated from resting spores, germinating spores, and appressoria of the soybean rust pathogen Phakopsora pachyrhizi. This information will be used for gene expression analysis that will enable the discovery of genes required by the pathogen to infect soybean. Under Objective 3, Sub-objective 3.A, we have successfully grown multiple wild relatives of the cultivated peanut to test for infection with T. frezzii and we are currently developing our inoculation protocols with cultivated peanut due to our limited reserves of T. frezzii spores. We screened advanced soybean breeding lines created by ARS scientists at Stoneville, Mississippi, for resistance to soybean rust. Resistant lines will be evaluated in field trials to evaluate performance under natural infection conditions and for yield tests to determine the most promising lines to be released. Under Sub-objective 3.B, mapping data for the soybean rust resistance gene Rpp7 and the soybean genome sequence of the susceptible soybean cultivar ‘Williams 82’ enabled the identification of at least seven candidate Rpp7 genes in the resistant soybean accession PI 605823. These partial sequences of the Rpp7 candidate genes were used to design virus-induced gene silencing vectors for loss-of-resistance assays. Preliminary results indicate that silencing of at least one of the Rpp7 candidate genes compromises resistance. Additionally, a bacterial artificial chromosome library was constructed to sequence the entire Rpp7 region. The identification of Rpp7 will facilitate the breeding of soybeans with durable resistance against soybean rust. Under Sub-objective 3.C, 91 samples of various cereal rust species were received on various hosts from foreign countries including Spain, Georgia, and Ethiopia under an international USDA-APHIS permit. Viable rust pathogen material was harvested, increased on wheat, barley and/or oat seedlings and preliminary virulence tests were conducted on the recovered samples. Rust spores and ethanol-killed infected wheat and barberry leaves from all samples were shipped to ARS researchers at the Cereal Disease Laboratory in St. Paul, Minnesota, for genotyping and wheat resistance screening. We have a high-quality genome assembly for H. vastatrix and have developed an annotation pipeline. We are currently waiting to receive phasing data, which will be needed to ensure this is genome is reference-quality. We have also received back the short-read sequence data from the 15 historical H. vastatrix accessions which we will map back to our H. vastatrix genome when it is complete.

Accomplishments
1. Gene for resistance to soybean rust disease identified. The U.S. is the second largest producer and exporter of soybeans, valued at over $30 billion annually. Soybean rust is a devastating disease that negatively impacts soybean production throughout the world. While most soybean germplasm is susceptible to the disease, seven genetic regions have been identified that provide resistance. To identify a resistance gene called Rpp3, scientists from ARS (Ames, Iowa and Frederick, Maryland), and Iowa State University sequenced candidate genes from two resistant soybean lines. Five Rpp3 candidate genes were identified in each resistant line, but only one was identical in both lines. Gene expression analysis and gene silencing of the candidate genes in the two resistant lines suggest that a single Rpp3 candidate gene, Rpp3C3, is responsible for Rpp3-mediated resistance. This research will allow soybean breeders to rapidly differentiate between known and potentially novel variants of Rpp3, facilitating the breeding of resistance.

U.S. DEPARTMENT OF AGRICULTURE

Foreign Disease-Weed Science Research: Frederick, MD