1a.Objectives (from AD-416):
The two objectives of the proposed research are (1) to develop molecular diagnostic assays to distinguish South American isolates of M. oryzae that infect wheat from native U.S. isolates of M. oryzae that are non-pathogenic on wheat and (2) to identify M. oryzae genes involved in pathogenesis and wheat genes involved in resistance. Specifically, we will (1) Perform whole genome sequencing of South American M. oryzae isolates infecting wheat and other related grasses; (2) perform whole genome sequencing on select isolates of M. oryzae collected in North America that do not infect wheat; (3) assemble and compare genomic sequences to identify unique regions; (4) develop and evaluate candidate genomic regions for diagnostic purposes, and (5) obtain and evaluate gene expression data from M. oryzae infected wheat.
1b.Approach (from AD-416):
Whole genome sequencing and post-sequencing comparisons will be used to rapidly identify unique loci between the M. oryzae pathotypes that infect wheat and the pathotypes that infect related grass species. This approach is made feasible by virtue of “next generation” sequencing platforms that enable fast and cost-effective whole genome sequencing projects. We have previously utilized this technology to sequence an isolate of wheat blast obtained from a 2011 outbreak of wheat blast in Bolivia. As part of this agreement, we will collect and prepare isolates of M. oryzae from North and South America collected from wheat and other grasses. Although the virulent pathotype of M. grisea (i.e. "South American wheat blast") has not been found outside of South America, it is important to sequence isolates collected from both continents in order to design a robust detection assay that is not prone to signaling false-positives. Additionally, RNA from wheat plants infected with M. oryzae will be used for deep sequencing to ascertain the genes involved in pathogenicity/resistance. Preparation of the M. oryzae isolates and isolation of high-quality genomic DNA and RNA will be performed at the FDWSRU at Ft. Detrick in the necessary containment laboratory/greenhouse facilities, and the sequencing will be performed by collaborators at Kansas State University. Post-sequencing analysis will be performed jointly between ARS and KSU, and final testing of the molecular diagnostic assays will be performed by ARS staff at Ft. Detrick.
Wheat blast, caused by the fungal pathogen Magnaporthe oryzae, is a potential threat to U.S. wheat production. Several distinct pathotypes of the pathogen have been identified, each with specificity towards a particular host. The objective of the proposed research is to develop molecular diagnostic assays to distinguish South American isolates of M. oryzae that infect wheat from native U.S. isolates of M. oryzae that do not cause disease on wheat. Two isolates of the pathogen recently collected in Bolivia, and one historic isolate collected in Brazil, have been used to isolate high-quality DNA for “next generation” sequencing. Genomic sequencing of these isolates has been completed, and these genome sequences are being compared to sequences obtained from U.S. isolates of M. oryzae. Additional isolates will be sequenced as necessary, and potential markers for use as diagnostic tools will be tested against a diverse collection of M. oryzae isolates.