Development of Multiplex RT-PCR for Simultaneous Detection of Garlic Viruses and the Incidence of Garlic Viral Disease in Garlic Genetic Resources

Abstract

Garlic generally becomes coinfected with several types of viruses belonging to the Potyvirus, Carlavirus, and Allexivirus genera. These viruses produce characteristically similar symptoms, they cannot be easily identified by electron microscopy (EM) or immunological detection methods, and they are currently widespread around the world, thereby affecting crop yields and crop quality adversely. For the early and reliable detection of garlic viruses, virus-specific sets of primers, including species-specific and genus-specific primers were designed. To effectively detect the twelve different types of garlic viruses, primer mixtures were tested and divided into two independent sets for multiplex polymerase chain reaction (PCR). The multiplex PCR assays were able to detect specific targets up to the similar dilution series with monoplex reverse transcription (RT)-PCR. Seventy-two field samples collected by the Gyeongbuk Agricultural Technology Administration were analyzed by multiplex RT-PCR. All seventy two samples were infected with at least one virus, and the coinfection rate was 78%. We conclude that the simultaneous detection system developed in this study can effectively detect and differentiate mixed viral infections in garlic.

A new virus found in garlic virus complex is a member of possible novel genus of the family Betaflexiviridae (order Tymovirales)

“…GYMaV as a component of garlic VCs should be considered in the development of virus-free garlic varieties. Many surveys of garlic viruses previously reported were based on target specific methods, since specific detection tools were utilized (Chen & Adams, 2001; Chen, Chen & Adams, 2002; Fajardo et al, 2001; Fayad-Andre, Dusi & Resende, 2011; Nam et al, 2015; Taglienti et al, 2018). Although this is the first report of GYMaV, we cannot rule out its presence on a larger geographical and temporal scale.…”

The unfolded protein response and programmed cell death are induced by expression of Garlic virus X p11 in Nicotiana benthamiana

“…Garlic virus X (GarVX), a member of the genus Allexivirus (family Alphaflexiviridae) shares a similar genomic organization with carlaviruses, potexviruses and foveaviruses, and is widely detected in Allium species worldwide (Adams et al, 2004;Chen et al, 2004;Nam et al, 2015;Song et al, 1998;Wylie et al, 2011Wylie et al, , 2014. Potexviruses, carlaviruses and foveaviruses have three genes, forming the triple gene block (TGB), that are involved in virus movement.…”

Multiplex RT-PCR identification of five viruses associated with the watermelon crops in the Brazilian Cerrado

“…Several researchers around the world developed methods with mRT-PCR to simultaneously identify and differentiate plant viruses for various crops, for instance the identification of pospiviroids in grapevine, with detection rate superior to 60% in four of five viroid samples analyzed (Hajizadeh et al, 2012), potexvirus, carlavirus, and tobamovirus in pepino, with 5% to 40% virus detection (Ge et al, 2013), potyvirus and polerovirus in sugarcane (Xie et al, 2009), cucumivirus and tobamovirus in tomato, with detection superior to 75% (Chen et al, 2011); potyvirus and allexivirus in onion (Kumar et al, 2010) and garlic (Nam et al, 2015), and crinivirus in tomato, lettuce (Lactuca sativa L), and melon (Cucumis melo L.) (Wintermantel and Hladky, 2010). Here, we detected all the five viruses tested (CMV, PRSV-W, WMV, ZLCV, and ZYMV) in almost all locales, except CMV in Lagoa da Confusão and ZYMV in Lagoa da Confusão and Gurupi.…”

“…Multiplex systems have been developed for the detection of multiple viral infections. Several genera have already been detected, for instance, astrovirus, rotavirus and reovirus (Jindal et al, 2012), carlavirus (Nam et al, 2015), crinivirus (Wintermantel and Hladky, 2010), nodavirus (Senapin et al, 2010), potyvirus, and allexivirus (Kumar et al, 2010), in animals (Jindal et al, 2012;Senapin et al, 2010) and plants (Kumar et al, 2010;Nam et al, 2015;Wintermantel and Hladky, 2010). According to the literature, the identification of viruses using mRT-PCR with specific primers has been widely used to detect viruses associated with different important crops around the world such as grapevine (Vitis vinifera) (Hajizadeh et al, 2012), pepino (Solanum muricatum) (Ge et al, 2013), potato (Solanum tuberosum L.) (Agindotan et al, 2007;Rigotti and Gugerli, 2007), Prunus spp (Jarosova and Kundu, 2010), sugarcane (Saccharum spp) (Xie et al, 2009) and tomato (Solanum lycopersicum L.) (Chen et al, 2011).…”