Development of detection method for Leifsonia xyli subsp. xyli, causal organism of ratoon stunting disease of sugarcane by quantitative polymerase chain reaction - high resolution melting analysis (qPCR-HRM)
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Abstract
Ratoon stunting disease (RSD) in sugarcane causes by Leifsonia xyli subsp. xyli (LXX) pathogen, leading to lower yields 5-30% and up to 50% in susceptible cultivars. Its symptoms are stunt, short internodes which are easily confused with other diseases or shown no symptom. Thus, sensitive and accurate detection method is needed. This study aims to develop a reliable detection method for this pathogen based on quantitative polymerase chain reaction (qPCR) and high resolution melting analysis (HRM) of qPCR product. Sugarcane samples in a total of 240 samples were collected from Kanchanaburi Sugarcane Research Station and Suphan Buri Field Crops Research Center. Genomic DNA was extracted from sugarcane stem and leaf for comparison on LXX detection by conventional PCR and qPCR-HRM techniques. The conventional PCR technique with two primer pairs, Pat1-F2/Pat-R2 which targeted Pat1 gene and CxxITSf#5/CxxITSr#5 which targeted the 16S-23S ribosomal DNA internal transcribed spacer (ITS) region, detected 29 and 61 of LXX positive samples, respectively. The qPCR-HRM method using Leifsonia-generic quantitative PCR primers LayF/LayS to target the ITS region detected 96 of LXX positive samples and became more efficient method. The qPCR-HRM product was further verified by having the same melting peak at 83.6 ºC as LXX and validated by DNA sequencing of ITS region (606 bp) matched to the ITS region of LXX strains from GenBank with 100% identity. The results revealed that qPCR-HRM is the most highly sensitive and accurate detection method for LXX.
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References
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