Construction of full-length infectious clones of turnip mosaic virus isolates infecting Perilla frutescens and genetic analysis of recently emerged strains in Korea

Abstract

Perilla is an annual herb with a unique aroma and taste that has been cultivated in Korea for hundreds of years. It has been widely cultivated in many Asian and European countries as a food and medicinal crop. Recently, several viruses have been reported to cause diseases in perilla in Korea, including turnip mosaic virus (TuMV), which is known as a brassica pathogen due to its significant damage to brassica crops. In this study, we determined the complete genome sequences of two new TuMV isolates originating from perilla in Korea. Full-length infectious cDNA clones of these two isolates were constructed, and their infectivity was tested by agroinfiltration of Nicotiana benthamiana and sap inoculation of Chinese cabbage and radish plants. In addition, we analyzed the phylogenetic relationship of six new Korean TuMV isolates to members of the four major groups. We also used RDP4 software to conduct recombination analysis of recent isolates from Korea, which provided new insight into the evolutionary relationships of Korean isolates of TuMV.

Fig. 1

(A) Results of PCR amplification of the full-length grenome of TuMV from perilla, with negative and positive controls. cDNA from a TuMV isolate that had previously been amplified successfully served as a positive control. (B) Colony PCR results of screening for full-length TuMV clones. (C) Confirmation of the positive colonies by digestion with ApaI and XmaI.

Fig. 2

(A) Symptoms induced by the TuMV isolates KPF-1 and KPF-2 on Nicotiana benthamiana at 7, 10, and 14 dpi. (B) Symptoms induced by the isolates KPF-1 and KPF-2 on radish cv. Iljin and Chinese cabbage cv. CR Victory at 14 dpi

Fig. 3

The positions of amino acid differences in the polyproteins of TuMV isolates KPF-1 and KPF-2

Fig. 4

A phylogenetic tree constructed by the maximum-likelihood method with 1000 bootstrap replicates, using MEGA 7.0, based on the complete genome nucleotide sequence of TuMV isolates from around the world. The genome sequence of potato virus Y was used to root the tree. Labels show the isolate name or abbreviation/GenBank accession number/original host (Table 2). The labels of the new Korean isolates from perilla (KPF-1, KPF-2), radish (KRS-3, KRS-8), and Chinese cabbage (KBC-1, KBC-8) are shown in bold font on pink bars.

Fig. 5

A phylogenetic tree constructed by the maximum-likelihood method with 1000 bootstrap replicates, using MEGA 7.0, based on the complete genome nucleotide sequence of Korean TuMV isolates. The genome sequence of potato virus Y was used to root the tree. Labels show the isolate name or abbreviation/GenBank accession number. For further details including the host plant, see Table 2. The labels of the new Korean isolates from perilla (KPF-1, KPF-2), radish (KRS-3, KRS-8), and Chinese cabbage (KBC-1, KBC-8) are shown in bold font.

Fig. 6

Recombination events identified using RDP4, supported by all seven detection methods (see Table 4). Recombination events 1 and 3 were detected in isolate BE, and event 2 was detected in isolates HJY1, HJY2, and R007.