A Miller, JVP Morales, MB Rollins, M Flasco and AB Gama
Louisiana State University Agricultural Center, Department of Plant Pathology and Crop Physiology, Baton Rouge, Louisiana, United States of America; agama@agcenter.lsu.edu
Systemic sugarcane diseases were silently introduced to new areas due to the lack of appropriate and specific detection methods. Two bacterial diseases are the focus of the Sugarcane Disease Detection Lab (SDDL) in Louisiana – ratoon stunt disease (RSD), caused by Leifsonia xyli subsp. xyli, and leaf scald (LS), caused by Xanthomonas albilineans. The SDDL also tests for Sugarcane yellow leaf virus (SCYLV), the causal agent of yellow leaf disease, as part of Louisiana’s seed cane certification program. More sensitive and specific techniques aligned with high-throughput nucleic acid extraction methods are desirable to improve detection and to facilitate statewide surveys on these diseases. Quantitative SYBR-based qPCR and RT-qPCR protocols were developed for the bacterial and viral pathogens, respectively. The sensitivity of the qPCR and RT-qPCR protocols was 100- and 10,000-fold higher than traditional PCR considering relative quantification. Two high-throughput DNA extraction methods and one RNA extraction method were tested for the pathogens and will facilitate statewide surveys for RSD and LS, while the optimized RNA extraction method with an additional DNAse step improved the quality of the samples. Improved quantification and detection methods will increase processing efficiency and can aid in understanding the distribution and epidemiology of these diseases.