First Report of a Mixed Infection of Tomato mottle mosaic virus and Tobacco mild green mosaic virus on Eggplants in China

Abstract

Eggplant (Solanum melongena L.) is an economically important vegetable crop in China. During March 2015 to June 2017, eggplant plants with typical viral symptoms, including dark purple mottle on flowers and slight mosaic and distortion on leaves, were observed at several farms in Shandong, Henan, Liaoning, and Hebei provinces, China. Visual observations estimated the symptom incidence at 20 to 40%. In total, 23 plants were sampled, and four samples (two flowers and two leaves) were randomly selected for total RNA extraction. High-throughput sequencing of small interfering RNA was performed with the pooled extracts using a second-generation sequencing technique by Igenecode Co. Ltd. (Beijing, China), and then the RNA of the virus was screened with bioinformatics methods. A sequence analysis revealed that 52.14 and 47.46% of the total virus sRNA was mapped to tomato mottle mosaic virus (ToMMV) and tobacco mild green mosaic virus (TMGMV) (NCBI reference sequences NC_022230.1 and NC_001556.1), respectively. To validate the presence of ToMMV and TMGMV, total RNA was extracted from each of the four samples, and reverse transcription polymerase chain reaction (RT-PCR) assays were carried out using primer pairs ToMMV-dF (5′-CTGGAGAAGACTGGGTCTAG-3′)/ToMMV-dR (5′-TTCGGTAAGTTCAATGGGACCT-3′) (Li et al. 2017) and 524-F (5′-CGTCATCGAGTACGTTTTAA-3′)/524-R (5′-AGGAAATCTCACAACAATAG-3′) (Cohen et al. 2001). Amplicons of 363-bp ToMMV CP and 500-bp TMGMV CP sequences were obtained from each of the four samples, and the sequences were 100% identical, respectively. Sequences of isolates QZ17091801 and QZ17102625 were deposited in GenBank (MH211033 for CP of ToMMV and MG807477 for CP of TMGMV). BLAST analysis showed that ToMMV CP sequence of the eggplant isolate shared 100% identity with that of a Chinese pepper isolate (KR824951.1) and a U.S. tomato isolate (KT810183.1), whereas the TMGMV CP sequence shared 99% identity with that of a German tobacco isolate (AJ429096.1) and a French trailing petunias isolate (AM262165.1). Field surveys were conducted in several provinces of north China, and 23 flower and leaf samples showing dark purple mottle and chlorotic symptoms were collected and tested by RT-PCR. ToMMV was detected in 20 of the 23 symptomatic samples, whereas TMGMV was detected in all of the 23 samples. To confirm that these eggplant plants were infected by ToMMV and TMGMV, one of the four samples was inoculated back to 10 healthy 3-week-old eggplant plants (cv. Heili) using mechanical inoculation. Ten days later, slight mosaic and distortion symptoms were observed on leaves of the inoculated plants. Total RNA was extracted from the symptomatic leaves and used as template for the ToMMV and TMGMV detection by RT-PCR. Both 363-bp ToMMV CP and 500-bp TMGMV CP sequences obtained were identical to that of the original isolate, confirming the inoculated eggplants were mix infected by ToMMV and TMGMV. To the best of our knowledge, this is the first record of a mixed infection of eggplant by ToMMV and TMGMV in China. Given the economic and nutritional importance of eggplant in China, the occurrence of both viruses on eggplant would be likely to negatively affect yield and farmers’ income. Therefore, ToMMV and TMGMV should be considered in future disease management of susceptible vegetable and ornamental plants.