Loop‐Mediated Isothermal Amplification Assays for Rapid Detection of Phytoplasma and Spiroplasma in Maize and in the Insect Vector Dalbulus maidis

Corn stunt disease complex (CSDC), caused by pathogens transmitted by the leafhopper Dalbulus maidis , is one of the most important maize diseases in South America. The agents include Spiroplasma kunkelii and ‘ Candidatus Phytoplasma asteris ’ strain MBS, both bacteria of the class Mollicutes. Traditional diagnostic approaches are mainly based on the visual assessment of plant symptoms and polymerase chain reaction (PCR). Loop‐mediated isothermal amplification (LAMP) emerges as a rapid, simple, specific and sensitive alternative for pathogen detection. This study aimed to develop LAMP‐based methodologies for detecting S. kunkelii and ‘ Ca . P. asteris ’ in maize and D. maidis . Three sets of LAMP primers were designed for each pathogen, and the most effective assays, F1 (targeting the 16S rRNA gene of ‘ Ca . P. asteris ’) and E2 (targeting the spiralin gene of S. kunkelii ), enabled efficient detection of the respective pathogens in maize and leafhoppers after incubation at 65°C for 45 min. At least 100 ‘ Ca . P. asteris ’ strain MBS or S. kunkelii ‐infected and uninfected samples obtained under controlled conditions were tested by PCR and compared with the LAMP assays. The LAMP methodologies showed high specificity and sensitivity, detecting up to 0.1 and 0.001 fg μL −1 of DNA for ‘ Ca . P. asteris ’ and S. kunkelii , respectively. In addition, a multiplex LAMP assay was developed to detect these mollicutes without differentiation. Crude extracts from plants or insects were also successfully used directly in the LAMP reaction, eliminating the DNA purification step. Altogether, LAMP‐based methodologies demonstrated to be a practical and effective alternative for the rapid detection of ‘ Ca . P. asteris ’ strain MBS and S. kunkelii .