Plant Pathol. J. 36(1) : 76-86 (2020) https://doi.org/10.5423/PPJ.OA.12.2019.0298 The Plant Pathology Journal pISSN 1598-2254 eISSN 2093-9280 ©The Korean Society of Plant Pathology Research Article Open Access Rapid and Sensitive Detection of Lettuce Necrotic Yellows Virus and Cucumber Mosaic Virus Infecting Lettuce (Lactuca sativa L.) by Reverse Transcription Loop-Mediated Isothermal Amplification Yubao Zhang 1, Zhongkui Xie1*, John D Fletcher2, Yajun Wang1, Ruoyu Wang1, Zhihong Guo1, and Yuhui He1 1Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences, Lanzhou 730000, China 2The New Zealand Institute for Plant and Food Research, PB 4704 Christchurch, New Zealand (Received on December 16, 2019; Accepted on January 10, 2020) Cucumber mosaic virus (CMV) is damaging to the for both RT-LAMP assays was greater by a factor of growth and quality of lettuce crops in Lanzhou, China. 100 compared to the conventional reverse transcription Recently, however, for the first time an isolate of lettuce polymerase chain reaction assays. This rapid, specific, necrotic yellows virus (LNYV) has been detected in let- and sensitive technique should be more widely applied tuce crops in China, and there is concern that this virus due to its low cost and minimal equipment require- may also pose a threat to lettuce production in China. ments. Consequently, there is a need to develop a rapid and efficient detection method to accurately identify LNYV Keywords : cucumber mosaic virus, lettuce, lettuce necrotic and CMV infections and help limit their spread. Re- yellows virus, RT-LAMP amplification, RT-PCR verse transcription loop-mediated isothermal amplifi- cation (RT-LAMP) assays were developed to detect the Handling Editor : Ho-Jong Ju nucleoprotein (N) and coat protein (CP) genes of LNYV and CMV, respectively. RT-LAMP amplification prod- Lettuce (Lactuca sativa L.) is the most economically im- ucts were visually assessed in reaction tubes separately portant vegetable crop belonging to the family Asteraceae using green fluorescence and gel electrophoresis. The (Krause-Sakate et al., 2001) and is typically eaten cold, assays successfully detected both viruses in infected raw, in salads, sandwiches, hamburgers, tacos as well as in plants without cross reactivity recorded from either many other dishes around the world (Fletcher et al., 2005; CMV or LNYV or four other related plant viruses. Op- Wikipedia, 2019). In China, the economic importance of timum LAMP reactions were conducted in betaine-free lettuce has also greatly increased during the past decade media with 6 mM Mg2+ at 65°C for LNYV and 60°C because of the rapid increase in demand for this crop, for 60 min for CMV, respectively. The detection limit which is now exceeding half the world’s production. How- was 3.5 pg/ml and 20 fg/ml using RT-LAMP for LNYV ever, lettuce is susceptible to a number of viral pathogens, and CMV plasmids, respectively. Detection sensitivity including Cucumber mosaic virus (genus: Cucumovirus; family: Bromoviridae). Cucumber mosaic virus (CMV) is *Corresponding author. one of the most widespread plant viruses with an extensive Phone) +86-931-4967204, FAX) +86-931-8273894 host range, infecting approximately 1,000 species in over E-mail) [email protected] ORCID 100 plant families that include cereals, fruits, vegetables Yubao Zhang and ornamentals, and can cause significant economic losses https://orcid.org/0000-0001-8438-0810 for numerous vegetable and horticultural crops (Roossinck, This is an Open Access article distributed under the terms of the 1999). In lettuce plants symptoms caused by CMV include Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0) which permits unrestricted plant stunting, leaf chlorosis, and mosaic as well as poor noncommercial use, distribution, and reproduction in any medium, head formation (Bruckart and Lorbeer, 1975; El-Borollosy provided the original work is properly cited. and Waziri, 2013). The virus is readily transmitted in a Articles can be freely viewed online at www.ppjonline.org. non-persistent manner by more than 75 species of aphids RT-LAMP Assays to Detect LNYV and CMV 77 (Palukaitis et al., 1992). It may also be transmitted mechan- immunosorbent assay (ELISA), reverse transcription (RT)– ically and can be seed-borne in a number of hosts. polymerase chain reaction (PCR) for LNYV and CMV In 2018, a preliminary field survey was conducted in (Callaghan and Dietzgen, 2005; Deyong et al., 2005; Dietz- a major lettuce cultivation area in Lanzhou City, Gansu gen and Francki, 1988; Koenig, 1981) and immunocapture Province, China. This survey detected lettuce necrotic yel- (IC)-RT-PCR for CMV (El-Borollosy and Waziri, 2013). lows virus (LNYV) for the first time in fields around Shi- These methods are valuable for identifying latent infec- chuan Town (Gaolan County, Lanzhou City, Gansu Prov- tions at most stages of infection. However, false negative ince, China; 36°10″N, 103°59″E). The main symptoms results and low sensitivity are common problems for the observed were plant stunting, leaf chlorosis, leaf roughness ELISA method. Moreover, PCR-based methods are expen- and occasional necrosis within leaf tissue. Mosaic symp- sive in terms of time and equipment and hence impractical toms were also present and attributed to a mixed LNYV for large-scale use. The IC-RT-PCR technique omits the infection of both CMV and lettuce mosaic virus (LMV) need to isolate RNA while providing a more rapid and (Fig. 1). LNYV is a member of the genus Cytorhabdovirus, less costly approach to prepare templates for amplification family Rhabdoviridae (Tordo et al., 2005). Infection by (Gambley et al., 2009); however, we have found IC-RT- members of the genus Cytorhabdoviridae is characterized PCR sensitivity insufficient in detecting low CMV concen- by the accumulation of enveloped virions in the cytoplasm trations (Zhang et al., 2017). of host cells (Jackson et al., 2005). LNYV causes a serious On the other hand, the loop-mediated isothermal amplifi- disease of lettuce crops grown in both Australia and New cation of nucleic acids (LAMP) assay, is not only sensitive Zealand, with the first reports published in 1963 and 1973, and relatively inexpensive to use but can also be routinely respectively (Fry et al., 1973; Stubbs and Grogan, 1963), conducted at a constant temperature between 58-70°C for and it continues to cause crop losses of up to 70% in some approximately 1 h in a water bath or on a heating block. cultivated areas (Fletcher et al., 2017; unpublished report). The technique can also be used for amplifying RNA by There also have been isolated reports of similar lettuce adding reverse transcriptase to the LAMP reaction mixture virus infections in Spain (Rubio-Huertos and Garcia-Hidal- (often referred to as RT-LAMP) (Bhat et al., 2013). Both go, 1982), Italy (Ragozzino et al., 1989), and Great Britain LAMP and RT-LAMP use four to six primers along with (Blancard et al., 2006). LNYV is transmitted in a persis- DNA polymerization with strand-displacing activity to tent, propagative manner by the blackcurrant-sowthistle generate amplification products. These products can be vi- aphid (Hyperomyzus lactucae L.) (Higgins et al., 2016). sually detected using agarose gel electrophoresis or through Ragozzino et al. (1989) also determined that the currant- color changes visualized by adding SYBR Green I, ethid- lettuce aphid (Nasonovia ribisnigri) was the primary vector ium bromide or calcein to the reaction mixture (Notomi et of LNYV disease outbreaks in Italy. al., 2000; Parida et al., 2008). Both methods have been suc- Viral disease management is dependent upon an accurate cessfully used in the detection of many DNA- (Bhat et al., detection procedure which is convenient, reproducible and 2013; Ravindran et al., 2012) and RNA-based (He et al., scalable for a wide range of samples (Viswanathan et al., 2016; Liu et al., 2010; Zhao et al., 2010) plant pathogens. 2013). Currently available methods commonly used for In this study, we developed and optimized a simple, rapid, detecting viruses that affect lettuce include enzyme-linked reliable and visual RT-LAMP method for the accurate de- Fig. 1. Healthy lettuce plant (A) and lettuce plant (B) infected with lettuce necrotic yellows virus (LNYV) exhibiting symptoms of plant stunting, leaf chlorosis, leaf roughness and intermittent necrosis within the leaf tissue. (C) Mosaic symptoms also present were attributed to the mixed infection of LNYV with both cucumber mosaic virus (CMV) and lettuce mosaic virus (LMV). 78 Zhang et al. tection of LNYV and CMV infections in lettuce plants. USA). Materials and Methods Cloning, sequencing, and sequence analysis. The first- strand viral cDNA was synthesized with the PrimeScript Plant sample collection and reference virus isolates. RT Reagent Kit with gDNA Eraser (Takara Biotechnology Within the cultivation areas of Shichuan and Donggang Co., Ltd., Dalian, China) using random hexamers. PCR towns, Lanzhou City, China, we collected naturally infect- was carried out in 12.5 μl reaction volumes containing 1.0 ed lettuce leaf samples that displayed typical viral symp- μl of the cDNA products, 1.25 μl of a 10× PCR buffer (100 toms (i.e., plant stunting, leaf chlorosis, leaf roughness and mmol/l Tris-HCl [pH 8.3], 500 mmol/l KCl, 15 mmol/ intermittent necrosis in leaf tissues). From field experience l MgCl2), 0.4 mmol of a dNTP mixture, 0.3 U of Taq gained in New Zealand and from employing known (tested) polymerase (TaKaRa Ex Taq, Takara) and 0.1 μM of each positives of viruses from New Zealand used here as refer- LNYV-F and LNYV-R (or CMV-F and CMV-R) primers ences (Fletcher et al., 2017; unpublished), lettuce leaves (Table 1). Both PCR amplification consisted of 35 cycles were tested using RT-PCR to confirm their respective virus at 94°C for 30 s, 52°C for 45 s and 72°C for 1 min.