Submitted to: Plant Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/23/2016
Publication Date: 7/27/2016
Publication URL: http://handle.nal.usda.gov/10113/62974
Citation: Ellison, M.A., Mcmahon, M.B., Bonde, M.R., Luster, D.G., Palmer, C.L. 2016. In situ hybridization for the detection of rust fungi in paraffin embedded plant tissue sections. Plant Methods. 12:37. DOI: 10.1186/s13007-016-0137-3..

Interpretive Summary: Rust fungi infect a wide range of plant species making them of particular interest to plant pathologists. In order to study the interactions between these important pathogenic fungi and their host plants, it is useful to be able to differentiate fungal tissue from plant tissue. Researchers from USDA-ARS Ft. Detrick, MD, the University of Pittsburgh and Rutgers University tested in situ hybridization (ISH) protocols to visualize pathogens infecting their hosts using samples from plant tissues infected with 3 different rust fungi. The results of these tests show that there is a high degree of specificity when using this technique, allowing for a clear distinction to be made between tissue of the fungal pathogen and the tissue of the plant it has infected. This protocol can be applied to pathogens from different genera of fungi, resulting in no background staining of plant tissue. The in situ hybridization protocol will be a useful addition to the methods and techniques used to localize and identify pathogenic fungi within plant tissues.

Technical Abstract: Rust fungi infect a wide range of plant species making them of particular interest to plant pathologists. In order to study the interactions between these important pathogenic fungi and their host plants it is useful to be able to differentiate fungal tissue from plant tissue. This can be accomplished using the in-situ hybridization (ISH) protocol described here. In order to validate this protocol reproducibility samples were tested from P. horiana, U. transversalis and P. pachyrhizi infected plant tissue along side healthy Chrysanthemum x morifolium, Gladiolus x hortulanus and Glycine max tissue samples. The results of these tests show that there is a high degree of specificity when using this technique allowing for a clear distinction to be made between tissue of the fungal pathogen and the tissue of the plant it has infected. The in situ hybridization protocol will be a useful addition to the methods and techniques used to localize and identify pathogenic fungi within plant tissues.