Location: Foreign Disease-Weed Science ResearchTitle: Specific detection of the wheat blast pathogen (Magnaporthe oryzae Triticum) by loop-mediated isothermal amplification Author
|Yasuhara-bell, Jarred - Kansas State University|
|Farman, Mark - University Of Kentucky|
|Valent, Barbara - Kansas State University|
|Stack, James - Kansas State University|
Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/6/2018
Publication Date: 10/15/2018
Citation: Yasuhara-Bell, J., Pedley, K.F., Farman, M., Valent, B., Stack, J.P. 2018. Specific detection of the wheat blast pathogen (Magnaporthe oryzae Triticum) by loop-mediated isothermal amplification. Plant Disease. 102:2550-2559. https://doi.org/10.1094/PDIS-03-18-0512-RE.
DOI: https://doi.org/10.1094/PDIS-03-18-0512-RE Interpretive Summary: Wheat blast is an economically important fungal disease of wheat caused by the Triticum pathotype of Magnaporthe oryzae. The pathogen can infect all above ground parts of the plant, but infection of the head is the most prevalent symptom and the one most commonly associated with the disease. Infected heads can easily be mistaken for the symptoms caused by other diseases. Currently, there is no diagnostic assay for the identification and detection of the wheat blast pathogen designed to be deployed for use in the field. The objective of this work was to develop a loop-mediated isothermal amplification (LAMP) detection protocol for the wheat blast pathogen. The assay was based upon a molecular marker, MoT3, that was previously shown to be unique to the Triticum pathtype of M. oryzae. The LAMP assay was tested using a diverse panel of M. oryzae isolates and a DNA extraction method suitable for in-field use. The results of this work demonstrate applicability of the assay for field surveillance of wheat blast, as well as identifying the disease in non-wheat species that may serve as a reservoir and/or source of inoculum for nearby wheat fields.
Technical Abstract: Wheat blast, caused by Magnaporthe oryzae Triticum (MoT) pathotype, is an economically important fungal disease of wheat. Wheat blast symptoms are similar to Fusarium head scab and can cause confusion in the field. Currently, no in-field diagnostic exists for MoT. Loop-mediated isothermal amplification (LAMP) primers were designed to target the PoT2 and MoT3 loci, previously shown specific for M. oryzae and MoT, respectively. Specificity was determined using 158 M. oryzae strains from collected from infected wheat and other grasses and representing geographic and temporal variation. Negative controls included 50 Fusarium spp. isolates. Sensitivity was assessed using 10-fold serial dilutions of MoT gDNA. PoT2- and MoT3-based assays showed high specificity for M. oryzae and MoT, respectively, and sensitivity to ~5 pg DNA per reaction. PoT2 and MoT3 assays were tested on MoT-infected wheat seed and spikes and identified M. oryzae and MoT, respectively, using a field DNA extraction kit and the portable Genie II system. The mitochondrial nad5 gene (NADH-dehydrogenase), an internal control for plant DNA, was multiplexed with PoT2 and MoT3 and showed comparable results to individual assays. These results show applicability for MoT field surveillance, as well as identifying non-wheat species that may serve as a reservoir and/or source of inoculum for nearby wheat fields.