Tomato (Solanum lycopersicum L.), an economically important vegetable, is widely grown around the world with leading tomato producing countries in China, the United States, Italy and Spain . Although most of tomatoes are field-grown, protected production systems (greenhouse) have increased significantly in recent years, including in North America . Pepino mosaic is one of several emerging viral diseases that seriously constrain the profitable production of tomatoes worldwide .
Pepino mosaic virus (PepMV) from the genus Potexvirus in the family Alphaflexiviridae was first reported to infect pepino (S. muricatum L.) in 1980 . This virus was not considered an issue until 20 years later when it was first reported to infect greenhouse tomatoes in the Netherlands . Since then, the disease has become widespread in Europe [6–14]. PepMV has also caused serious problems on greenhouse tomatoes in North America [15–18] and in South America [19–21]. Furthermore, this virus was detected in China , Syria , and South Africa . Thus, PepMV has become one of the most economically important viruses infecting greenhouse tomatoes worldwide [3, 25, 26]. The typical disease symptoms on tomatoes include mosaic, yellow patches, necrotic lesions and uneven ripening of fruits resulting in marbling or flaming in appearance on mature fruits. It was estimated that the poor quality of PepMV-infected fruits could reduce tomato market value by up to 36% .
Three major genotypes of PepMV (namely EU, US1 and CH2), which share only 78-82% genomic nucleotide sequence identity, have been reported [9, 10, 16, 21, 25, 26]. Even though a greater level of PepMV genetic diversity was initially recognized in the American continents [16, 21], further population genetic analysis in Canada and the U.S. showed a predominant EU genotype, with only a small fraction of field isolates in US1 and CH2 genotypes . However, several studies in Europe in recent years demonstrated that the prevalent PepMV genotype has shifted from a predominant EU [11, 28] to CH2 [14, 29–31]. Recent outbreaks of pepino disease in association with the CH2 genotype in broader geographic regions in the world, including the Middle East  and South Africa , raised concerns of possible seed transmission. Although PepMV on tomato is localized on seed coat (testa) and not in embryo, mechanical transmission from a contaminated seed could easily induce a new infection . A low rate (0.026%) of PepMV seed transmission in tomato has already been observed . These results on seed transmission demonstrated the importance of selecting and planting tomatoes from PepMV-tested negative seed lots. With intensive cultural practices, as required under a greenhouse tomato production system (i.e., grafting, de-leafing, inter-cropping and bumble bee pollination), even with a small number of PepMV-infected plants serving as sources of initial inocula could result in a serious disease epidemic. Thus, a timely detection and rapid genotype determination would be a prerequisite for deploying effective strategies in disease management.
Currently, several molecular-based methods have been developed to determine the genetic diversity of PepMV, including restriction fragment length polymorphism (RFLP) [34, 35], reverse transcription- polymerase chain reaction (RT-PCR) and sequencing [14, 17], real-time RT-PCR , and hybridization . All such molecular methods required some specialized instruments or sophisticated laboratory conditions, making it difficult to apply for field analysis. With recent trends in genotype shifting in Europe, growers in North America are concerned about their crop’s health status and would request additional sequencing to determine the specific genotype . Although this method is accurate, it is costly and time consuming. Thus, we were interested in developing a rather simple and quick reverse transcription loop-mediated isothermal amplification (RT-LAMP) for efficient identification of PepMV genotypes. In recent years, the RT-LAMP technology [37, 38] has been successfully applied for plant virus and viroid detection [39–47]. In an effort to understand the current genetic diversity and evolution of PepMV in North America, we established a surveillance system with assistance from participating growers in Canada, Mexico and the U.S.A. on sample collection. We developed and used LAMP to monitor the presence and distribution of PepMV genetic diversity in North America.