Development and Evaluation of a Loop-Mediated Isothermal Amplifcation (LAMP) Assay for Specific and Sensitive Detection of Puccinia melanocephala Causing Brown Rust in Sugarcane

Abstract

Sugarcane brown rust (SCBR), caused by Puccinia melanocephala, is a destructive fungal disease that has extensively spread in the sugarcane-cultivating regions across the world. Early monitoring plays an important role in predicting the P. melanocephala epidemic and managing SCBR. However, accurately identifying SCBR based on symptoms and urediniospore morphology at the initial stage is a challenge. Further, it is tedious, time-consuming, labor-intensive, and requires expensive equipment to detect P. melanocephala using PCR-based methods. Loop-mediated isothermal amplification (LAMP) technology is renowned for its speed, simplicity, and low equipment requirements for specifically and sensitively identifying many pathogens. Therefore, in this study, a novel and highly sensitive LAMP assay was developed for the specific detection of P. melanocephala in sugarcane. Here, the internal transcribed spacer (ITS) sequence of P. melanocephala was selected as the target gene for LAMP primer design. Based on the color change of SYBR Green I and gel electrophoresis, specific LAMP primers were screened. Further, the optimal reaction conditions for the LAMP assay were determined at 63 °C for 60 min. The LAMP assay showed a high degree of specificity for the detection of P. melanocephala in sugarcane, with no cross-reactivity with other fungal pathogens. The established LAMP protocol was highly sensitive and can be used to detect as low as 1 pg/μL of P. melanocephala plasmid DNA, which is comparable to that of nested PCR and ~100 times more sensitive than conventional PCR. Finally, the detection rate of the LAMP method was higher than that of conventional and nested PCR in field samples.