Location: Sugarcane ResearchTitle: Detection of Puccinia kuehnii causing sugarcane orange rust with a loop-mediated isothermal amplification-based assay Author
|Chandra, Amaresh - Indian Institute Of Sugarcane Research|
Submitted to: Molecular Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/8/2016
Publication Date: 2/1/2016
Publication URL: http://handle.nal.usda.gov/10113/63017
Citation: Chandra, A., Keizerweerd, A.T., Grisham, M.P. 2016. Detection of Puccinia kuehnii causing sugarcane orange rust with a loop-mediated isothermal amplification-based assay. Molecular Biotechnology. 58:188-196.
Interpretive Summary: Orange rust was first observed in the U.S. domestic sugarcane industry in Florida in 2007 and a few years later in Louisiana in 2012. In the early stages of development, the symptoms of orange rust are very similar to those caused by brown rust. The two rust diseases are caused by different species of the fungal genus, Puccinia. To correctly identify orange rust, a LAMP (loop-mediated isothermic applification)-based assay was developed. Although several laboratory assays are available to correctly identify orange rust, the LAMP assay is more economical and easier to perform. The assay is performed in a single reaction tube at a constant temperature in contrast to other assays that require cycling the reaction mixture through multiple temperatures requiring expensive instruments to precisely control the temperature. A visual color change in the reaction mixture in the tube occurs when the orange rust fungus is present. Other assay protocols require a post-reaction step to determine if the results are positive or negative for the orange rust fungus. The LAMP assay is highly specific and sensitive and because of the simplicity of the protocol can be adapted to field as well as laboratory use.
Technical Abstract: Puccinia kuehnii is a fungal pathogen that causes orange rust in sugarcane, which is now prevalent in many countries including Florida and Louisiana in the U.S. At the early stage of the disease, it is almost indistinguishable from brown rust, which is caused by another species of Puccinia, viz., Puccinia melanocephala. Though several PCR assays are available to detect these diseases, the loop-mediated isothermal amplification (LAMP)-based assay has been reported to be more economical and easier to perform. The present study was undertaken to detect P. kuehnii genomic DNA, isolated both from pure culture as well as infected tissues, using four sets of specific primers corresponding to a unique DNA sequence of P. kuehnii. Under isothermal conditions, DNA is amplified with high specificity, efficiency, and rapidity. Moreover, visual judgment of color change in about 1 hr without further post-amplification processing makes the method convenient and economical. Specificity of the LAMP test was checked with DNA of P. melanocephala. No reaction was found with P. melanocephala. Sensitivity of the LAMP method was observed to be ten times higher than that of RT-PCR.