Development of PCR and loop mediated isothermal amplification assay for the detection of bakanae pathogen Fusarium fujikuroi

Bakanae caused by Fusarium fujikuroi (Nirenberg) is an emerging disease of rice in India. Although, two other pathogens are closely associated with the disease (F. proliferatum and F. verticillioides), F. fujikuroi is considered the major pathogen of this disease. Correct diagnosis and identification of the pathogen is the key to the successful management of plant diseases. PCR and loop mediated isothermal amplification (LAMP) based assays were developed for the detection of F. fujikuroi. Based on the unique sequences (NRPS31 gene) of F. fujikuroi obtained from GenBank accession KY953210, a specific marker BMFf1900 was developed for the amplification of F. fujikuroi with a sensitivity of 10 pg. For precise quantification of the inoculum, real time primers (NRPS31RT-F and NRPS31RT-R) were designed and sensitivity of the assay was increased to 10 fg. For the visualization of results under isothermal conditions, the LAMP assay was standardized. These PCR and LAMP based assays have been validated under field conditions, seed testing and in resistance evaluation of rice genotypes. The diagnostics developed are efficient, rapid and sensitive for the detection of F. fujikuroi which would be useful for testing seed health, validating varietal resistance and monitoring the pathogen inoculum.