Cyclone-based spore trapping, quantitative real-time polymerase chain reaction and high resolution melting analysis for monitoring airborne inoculum of Magnaporthe oryzae

Abstract

Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases in rice worldwide. We aimed to develop an integrated approach for convenient collection, quantification and characterisation of M. oryzae spores (airborne inoculum) in the field. We developed an easy-to-use cyclone-based spore trap (the AirSampler) and a standard procedure for handling a small amount of airborne spores. Using a specific primer pair or a probe designed for the single-copy gene mif23, SYBR Green and TaqMan assays could quantify 10 and 4 copy numbers, respectively, of M. oryzae DNA. During 2012 and 2013, the AirSampler and SYBR Green quantitative real-time polymerase chain reaction were used to monitor temporal dynamics of M. oryzae spores in nursery fields of rice showing symptoms of blast disease. During four cropping seasons, the new techniques could detect M. oryzae spores before the appearance of rice blast symptoms. The amount of spores was low in the early season, then increased, with high fluctuations during the mid-season and decreased to low levels at the heading stage in the late season. To improve the handling and storage of spore samples, we tested the effect of different treatments on the preservation of spore DNA. DNA loss was reduced with samples protected from ultraviolet B radiation, suspended in CTAB buffer, kept at room temperature or 4 degrees C and used for DNA extraction in 2 weeks. Finally, we demonstrated that the high resolution melting analysis could be used for rapid determination of A, D, A+D and C alleles of the avirulence gene pex31 (Avr-Pik/kp/km) in M. oryzae.