Detection of Fomitiporia torreyae and Fulviformes umbrinellus by multiplex loop‐mediated isothermal amplification ( mLAMP ) for diagnosis of Japanese pear dwarf

Japanese pear dwarf, caused by the fungi Fomitiporia torreyae or Fulviformes umbrinellus, is one of the most important diseases affecting Japanese pear ( Pyrus pyrifolia var. culta). To diagnose this disease, a multiplex loop‐mediated isothermal amplification ( mLAMP ) reaction using primer sets designed from the rDNA internal transcribed spacer ( ITS ) sequences of F. torreyae and F. umbrinellus was developed. The optimal conditions for simultaneous detection of the two pathogens were investigated. The best results were obtained at a reaction temperature of 65°C and a primer ratio of 1:1.5 ( F. torreyae  :  F. umbrinellus ). Fluorescently labelled mLAMP amplicons were precipitated using polyethyleneimine. As a result, multiplex detection was enabled by the fluorescent colour of precipitate under ultraviolet light. The detection limit of mLAMP was 100 fg of genomic DNA , which was 10 times more sensitive than the polymerase chain reaction ( PCR ) method. The mLAMP assay was applied to Japanese pear trunk samples from Aichi Prefecture, Japan, and the results were compared with those obtained using PCR . As a result, mLAMP was observed to be effective for the specific detection of F. torreyae or F. umbrinellus .