DEVELOPMENT OF A RAPID PCR NUCLEIC ACID LATERAL FLOW IMMUNOASSAY (PCR-NALFIA) BASED ON rDNA IGS SEQUENCE ANALYSIS FOR THE DETECTION OF MACROPHOMINA PHASEOLINA IN SOILS

Macrophomina phaseolina is a soil- and seed-borne generalist fungal pathogen with a global distribution and can infect more than 500 plant species. In infected tissues the fungus produces microsclerotia which enable it to survive in soil for 2-15 years, act as primary inoculum source and are needed for the correct identification of the pathogen. The disease occurrence and severity are directly related to the population of viable microsclerotia in soil. The ‘Nucleic Acid Lateral Flow Immunoassay’ using a generic ‘Lateral Flow Device’, combined with PCR employing labelled primers to detect a specific amplicon, enables to circumvent the use of electrophoresis, making the diagnostic procedure faster and easier. This study describes the development of the species-specific primers MP102F/ MP102R for M. phaseolina based on the intergenic spacer (IGS) of the rDNA sequence analysis. The primer specificity was checked and confirmed using 20 isolates of the pathogen and other 16 nontarget fungi. Microsclerotia of M. phaseolina (1, 10, 100 and 200) were manipulated under a stereomicroscope and their DNA was extracted from microsclerotia alone or mixed with different types of soil. The resulting DNA, used in the PCR-NALFIA assay, provided positive results for all the samples tested. A semi-quantitative greyscale reference card was developed using intervals corresponding to microsclerotia soil number. For this purpose, the normalized pixel grey volumes obtained after a densitometric analysis of the test line intensity generated by the LFD strips were used. Patent application relating to the method here described is pending