Detection of a wide range of viruses and viroids collected in Nara Prefecture fields by mRNA-Seq and evaluation of the detection system

Detection of viruses and viroids is mainly conducted by RT-PCR using specific primers for the identification of plant pathogens. However, it is difficult and time-consuming to detect pathogenic viruses using these methods for new strains, unexpected species, or infection of new host plants; in these circumstances, different methods need to be employed. Messenger RNA sequencing technology (mRNA-Seq) detects a wide range of viruses and viroids in a single experiment. Here, we describe a model case in which we detected RNA viruses, DNA viruses, and viroids collected from Nara Prefecture fields using mRNA-Seq and developed an RNA extraction method and data analysis for the establishment of this system. The RNA extraction method was optimized to improve the quality of RNA in chrysanthemum plant tissue. The reduction of the plant tissue in the RNA extraction buffer resulted in the improvement of RNA yields and quality. By introducing the criteria for read number and genome coverage, we detected RNA viruses (cucumber mosaic virus (CMV), tomato spotted wilt virus (TSWV)) and viroids (chrysanthemum stunt viroid (CSVd), chrysanthemum chlorotic mottle viroid (CChMVd)), but not DNA viruses (tomato yellow curl leaf virus (TYLCV), dahlia mosaic virus (DMV)). In addition to the read number and the genome coverage, the average nucleotide sequence identity of reads detected by BLAST homology search and the average length of the identical region are important for judging whether the virus and viroid have infected the plants. These results indicate that the mRNA-seq could detect a wide range of RNA viruses and viroids in plants.