Rapid Diagnosis of Soybean Seedling Blight Caused by Rhizoctonia solani and Soybean Charcoal Rot Caused by Macrophomina phaseolina Using LAMP Assays.

A new method of direct detection of pathogenic fungi in infected soybean tissues has been reported here. The method rapidly diagnoses soybean seedling blight caused by Rhizoctonia solani and soybean charcoal rot caused by Macrophomina phaseolina, and features loop-mediated isothermal amplification (LAMP). The primers were designed and screened using internal transcribed spacers (ITS) as target DNAs of both pathogens. An ITS-Rs-LAMP assay for R. solani and an ITS-Mp-LAMP assay for M. phaseolina that can detect the pathogen in diseased soybean tissues in the field have been developed. Both LAMP assays efficiently amplified the target genes over 60 min at 62°C. A yellow-green color (visible to the naked eye) or intense green fluorescence (visible under ultraviolet light) was only observed in the presence of R. solani or M. phaseolina after addition of SYBR Green I. The detection limit of the ITS-Rs-LAMP assay was 10 pg µl⁻¹ of genomic DNA; and that of the ITS-Mp-LAMP assay was 100 pg µl⁻¹ of genomic DNA. Using the two assays described here, we successfully and rapidly diagnosed suspect diseased soybean samples collected in the field from Jiangsu and Anhui provinces.