Plant Pathol. J. 33(2) : 184-192 (2017) https://doi.org/10.5423/PPJ.OA.10.2016.0224 The Plant Pathology Journal pISSN 1598-2254 eISSN 2093-9280 ©The Korean Society of Plant Pathology Research Article Open Access Development and Evaluation of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Tylenchulus semipenetrans Using DNA Extracted from Soil Zhi-Qiang Song1,2†, Ju-E Cheng2†, Fei-Xue Cheng2*, De-Yong Zhang1,2, and Yong Liu1,2* 1College of Plant Protection, Hunan Agricultural University, Changsha 410128, P. R. China 2Institute of Plant Protection, Hunan Academy of Agricultural Sciences, Changsha 410125, P. R. China (Received on October 20, 2016; Revised on December 13, 2016; Accepted on December 29, 2016) Tylenchulus semipenetrans is an important and wide- tive, specific and accurate technique for detection of T. spread plant-parasitic nematode of citrus worldwide semipenetrans in field soil, and contributes to the effec- and can cause citrus slow decline disease leading to tive management of citrus slow decline disease. significant reduction in tree growth and yield. Rapid and accurate detection of T. semipenetrans in soil is Keywords : citrus slow decline disease, DNA extraction, important for the disease forecasting and manage- loop-mediated isothermal amplification, nematode detec- ment. In this study, a loop-mediated isothermal am- tion, Tylenchulus semipenetrans plification (LAMP) assay was developed to detect T. semipenetrans using DNA extracted from soil. A set Handling Associate Editor : Kim, Ki Woo of five primers was designed from the internal tran- scribed spacer region (ITS1) of rDNA, and was highly specific to T. semipenetrans. The LAMP reaction was The citrus nematode, Tylenchulus semipenetrans Cobb performed at 63°C for 60 min. The LAMP product 1913, is an important plant-parasitic nematode that is was visualized directly in one reaction tube by adding widely distributed in citrus-growing regions worldwide SYBR Green I. The detection limit of the LAMP as- and causes citrus slow decline disease (El-Borai et al., say was 10–2 J2/0.5 g of soil, which was 10 times more 2002). T. semipenetrans is a sedentary semiendoparasite, sensitive than conventional PCR (10–1 J2/0.5 g of soil). and can infect all Citrus species and most hybrids of cit- Examination of 24 field soil samples revealed that the rus in the Rutaceae family (Verdejo-Lucas et al., 2000). LAMP assay was applicable to a range of soils infested Aboveground symptoms of the disease are stunting, naturally with T. semipenetrans, and the total assay slow growth, leaf chlorosis and abscission, reduced fruit time was less than 2.5 h. These results indicated that size and yield (Verdejo-Lucas and McKenry, 2004). Ad- the developed LAMP assay is a simple, rapid, sensi- ditionally, the wounds in citrus roots caused by T. semi- penetrans are subject to the invasion of plant pathogenic †These authors contributed equally to this work as first authors. fungi and/or bacteria leading to more severe damage to *Co-corresponding authors. citrus trees (Duncan, 2009). Yield losses caused by T. Y Liu semipenetrans are generally 10% to 30% (Verdejo-Lucas Phone) +86-731-84696568, FAX) +86-731-84696578 and McKenry, 2004). The diagnosis of the disease is very E-mail) [email protected] difficult as field symptoms are similar to physiological FX Cheng diseases or other plant diseases. In particular, etiolation Phone) +86-731-84696638, FAX) +86-731-84696578 symptoms of the disease were often misdiagnosed as cit- E-mail) [email protected] cc This is an Open Access article distributed under the terms of the rus Huanglongbing, and these diseased citrus trees were Creative Commons Attribution Non-Commercial License (http:// felled or eradicated leading to significant economic losses creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non- in some citrus orchards in China (Song et al., 2016). Thus, commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. a rapid and reliable detection method for T. semipenetrans becomes crucially important for effective control of this Articles can be freely viewed online at www.ppjonline.org. Rapid Detection of Tylenchulus semipenetrans in Soil by LAMP 185 nematode. semipenetrans (Lin et al., 2016). Traditional method for T. semipenetrans identification Damage to citrus caused by T. semipenetrans is related depends mainly on morphological observation of mature to population densities of J2 in the soil (Irshad et al., females, males and second-stage juveniles (J2) by micros- 2012). Detecting the presence of T. semipenetrans in the copy (Inserra et al., 1988; Rashidifard et al., 2015), which soil is essential for both the diagnosis of the disease and is time-consuming, laborious and requires taxonomic many cropping decisions, especially for establishing a expertise. In particular, it is difficult to distinguish J2 of cit rus nursery site (Liu et al., 2011). Although PCR-based T. semipenetrans from many other plant-parasitic and me thods described above identified J2 of T. semipen- non-plant-parasitic nematode species microscopically. etrans, they were designed to use DNA extracted from As an alternative strategy for T. semipenetrans identifica- isolated individuals that requires a long operation time tion, molecular diagnostic methods based on polymerase and experienced personnel (Liu et al., 2011; Maafi et al., chain reaction (PCR) have been developed and applied, 2012; Rashidifard et al., 2015). Hence, a rapid, simple including PCR-restriction fragment length polymorphism and time-saving nematode extraction method is also (RFLP) of the internal transcribed spacer region (ITS) needed for quick diagnostic of T. semipenetrans. The de- (Maafi et al., 2012; Park et al., 2009) and PCR with spe- velopment of soil DNA extraction methods has overcome cies-specific primer sets designed from rDNA-ITS (Liu et such problems, and these methods have been applied to al., 2011; Maafi et al., 2012). While PCR-based detection detect plant-parasitic nematodes in soil, such as Prat- methods provided faster, more reliable and more sensi- ylenchus spp. (Yan et al., 2008), Meloidogyne incognita, tive tools for T. semipenetrans identification when com- Heterodera glycines, and Globodera rostochiensis (Min pared with traditional morphological method, they are not et al., 2012). generally available for routine practical applications at Although a LAMP detection method of T. semipen- grassroots quarantine stations and plant protection orga- etrans was developed (Lin et al., 2016), the detection nizations because expensive and sophisticated laboratory sensitivity was relatively lower. The objectives of this equipment and skilled technicians are needed. Therefore, study were to develop a rapid, simple, specific and highly the development of rapid, simple and cost-effective detec- sensitive LAMP method for detection of T. semipenetrans tion methods is still needed for the specific diagnosis of T. directly from soil, and to confirm whether the method was semipenetrans. applicable to a wide range of soils naturally infested with Loop-mediated isothermal amplification (LAMP) is a T. semipenetrans. In particular, the specificity, sensitiv- sensitive and rapid nucleic acid amplification technology ity and field application of the method for detection of T. developed by Notomi et al. (2000) which can amplify semipenetrans were assessed. DNA under isothermal conditions (60–65°C) in less than an hour. The technique requires Bst DNA polymerase Materials and Methods with strand-displacement activity and a set of four to six specially designed primers that recognize six to eight dis- Nematode populations. All nematode populations used tinct sequences on the target DNA (Nagamine et al., 2002; in this study were listed in Table 1. T. semipenetrans was Notomi et al., 2000). The amplification products are stem- isolated from citrus rhizosphere soil in Yongzhou City, loop DNA structures with several inverted repeats of the Hunan Province, China, and was maintained on a suscep- target and cauliflower-like structures with multiple loops. tible mandarin orange planted in a plastic pot in a green- LAMP products can be confirmed with the naked eye by house. All nematodes had been identified by morphologi- adding a fluorescent DNA intercalating dye (e.g., SYBR cal characteristics and molecular diagnoses. Green I or propidium iodide) or a metal-ion indicator (e.g., calcein or hydroxynaphthol blue) to the reaction tube and DNA extraction. Two methods of DNA extraction were observing the color of the solution (Goto et al., 2009; Hill used in this study. Total genomic DNA was extracted et al., 2008; Iwamoto et al., 2003; Tomita et al., 2008). from soil using the FastDNA SPIN Kit for Soil (MP Bio- All LAMP steps, from amplification to detection, are medicals, Solon, OH, USA) according to the manufac- conducted in one reaction tube, and only a water bath or turer’s instructions, and dissolved to a final volume of 20 heating block is required, which make it suitable for both μl. DNA of all nematodes in Table 1 was extracted from a field- and lab-based pathogen detection. To date, LAMP single nematode following the method described by Htay has been successfully developed to detect several plant- et al. (2016), with some modifications. A single nematode parasitic nematodes including Bursaphelenchus xylophi- was hand-picked using a fine teasing needle and put into a lus (Kikuchi et al., 2009), Meloidogyne spp. (Niu et al., 0.2 ml sterile PCR tube containing 8 μl of distilled water 2011, 2012), Radopholus similis (Peng et al., 2012) and T. and 1 μl of 10× PCR buffer (Mg2+ free) (Takara, Dalian, 186 Song et al. Table 1. Species of plant nematode used to evaluate specificity of loop-mediated isothermal amplification (LAMP) assay No. Species Host plant Origin 1 Tylenchulus semipenetrans Citrus YongZhou, Hunan 2 Meloidogyne hapla Citrus YongZhou, Hunan 3 Pratylenchus coffeae Citrus YongZhou, Hunan 4 Helicotylenchus dihystera Citrus YongZhou, Hunan 5 Aphelenchus avenae Citrus YongZhou, Hunan 6 Filenchus spp.