A. Suwanngam Validation of LAMP assay for specific-detection of M.mali in Belgium GHENT UNIVERSITY FACULTY OF SCIENCE DEPARTMENT OF BIOLOGY ACADEMIC YEAR 2017-2018 Validation of The Novel Loop-mediated Isothermal Amplification (LAMP) for Specific- detection of Meloidogyne mali in Belgium ARUNEE SUWANNGAM Promoter: Prof. dr. ir. Wim Wesemael A thesis submitted to Ghent University in partial fulfilment of the requirements for the degree of International Master of Science in Agro- and Environmental Nematology 1 A. Suwanngam Validation of LAMP assay for specific-detection of M.mali in Belgium Validation of The Novel Loop-mediated Isothermal Amplification (LAMP) for Specific-detection of Meloidogyne mali in Belgium Arunee Suwanngam¹ ¹Ghent University, Department of Biology, Ledeganckstraat 35, 9000 Ghent, Belgium Abstract. The root-knot nematode, Meloidogyne mali has recently been added to the EPPO A2 list as a quarantine pest. This nematode cause damage to various economically important crops and the deterioration of woody plants in different geographical regions. To imply effective phytosanitary measure, control and prevention, fast and accurate diagnostics methods are required. The Mma-specific detection assays described by Zhou et al in 2017 were validated and optimized. The results showed that the specific PCR assay could detect as low as 36 pg of genomic DNA and corresponded with the detection limits of the LAMP reaction under optimized conditions. The specificity of both PCR and LAMP assay was high, without cross- amplification reaction with other related nematode species. Notably, the LAMP reaction successfully detected M. mali in 40 minutes. This assay was also adapted for on-site detection using the OptiGene plant lysis buffer to replace the full nucleic acid extraction method. In April 2018, 10 root samples (Ulmaceae) were collected from elm trees at the INBO research station where in the 1970s and 1990s possible infected elm trees from the Netherlands were planted. The results revealed that the ten samples were greatly amplified by PCR and LAMP assay. Moreover, these diagnosis results were confirmed by nucleotide sequences that shared 99-100% similarity with accession number KM887145, KM887146, KY433449 and KM433450 respectively. This analysis confirmed the presence of M. mali in Belgium 30 to 40 years after the introduction with infected elm trees. The Mma-specific detection assay offered high specificity, sensitivity and simplicity for rapid detection of Meloidogyne mali for further reliable pest risk analysis and assessment. Keywords: Meloidogyne mali, quarantine nematode, LAMP 2 A. Suwanngam Validation of LAMP assay for specific-detection of M.mali in Belgium INTRODUCTION Meloidogyne species are one of the prominent plant-parasitic nematodes that cause damage to the most important economic plants. They have a wide range of habitats, such as rivers and deserts. Meloidogyne mali, apple root-knot nematode causes significant damage to different kind of plants, especially apple and mulberry trees by stimulating gall-like swellings. This nematode is known as polyphagous with various host plants such as shrubs, perennials or herbaceous plants and particularly Ulmaceae and Rosaceae. Meloidogyne mali was first found in the northern part of Japan (Itoh et al., 1969). It was officially reported in several continents and countries including Italy, the Netherlands, France and United States of America. In addition, Meloidogyne mali has been defined as a quarantine pest in the EPPO A2 list (EPPO, 2017). In 1969, Meloidogyne mali was first reported in apple trees in Nagano Prefecture and its distribution was limited to the northern part of Japan (Itoh et al., 1969). Otherwise in 1984, it was abundantly found in the mulberry orchards in Hokkaido and Honshu province (Toida and Yaegashi, 1984). Later in 1988, it was found again in Kyushu island in the southern part of Japan. The epidemic in these areas may have been caused by the introduction of infected mulberry seedlings from other provinces of Japan (Araki, 1988). For the intercontinental spread to Europe, it has been estimated that Meloidogyne mali contaminated plant material from Asia was exported prior to the World War II. The plant material was used in the breeding program in the Netherlands for the Dutch Elm Disease. In 1979, this nematode was reported for the first time in Italy from the roots of the elm trees at San Rossore ( Pisa, Toscana region) and Mantignano (Firenze, Toscana region). From 2008-2014, it was widespread found in several areas in the Netherlands such as Baarn, Wageningen (Dutch Elm Disease Experimental Fields 3 A. Suwanngam Validation of LAMP assay for specific-detection of M.mali in Belgium on Ulmus), The Hague (on street trees) and Haarlem district (NPPO of the Netherlands, 2014; de Nijs et al., 2016). In France, this nematode was firstly identified in 2014-2015 at the nursey of INRA Nancy (Champenoux North-Eastern) and later in 2016 at Champenoux and Vincennes forest (Ile-de-France region) (NPPO of France, 2016). Most recently, it was found in New York, USA (EPPO, 2017; Eisenback et al., 2017). The epidemic of Meloidogyne mali in these countries was estimated that it was introduced by importing infected Elm trees from the Netherlands. Elm trees are important for the traditional rural landscape due to its highly tolerance to severe environmental conditions. Additionally, the elm trees are alternatively being used as ornamental plants, roadside trees and natural windbreaks or shelterbelts to prevent the damage caused by wind. This could be the reason why the elm trees have been planted in several territories of European countries (Harwood et al, 2011; Caudullo and Rigo, 2016). Under those circumstances, EPPO reported the possibility of Meloidogyne mali distribution in various countries such as Belgium, Denmark, France, Germany, Ireland, Italy, Spain, Slovakia, Romania and England by importing young elm trees from the original outbreak site in the Netherlands under the breeding program to increase the resistance of Elm trees against Dutch Elm Disease (Ahmed et al., 2013). The status of Meloidogyne mali in Belgium will be assessed in 2018. The pathogenesis of Meloidogyne mali starts after penetration of the second stage juvenile ( J2) into the roots and begins to feed on the host tissues, which is conducive to encouraging adjacent cells be separated and developed into specialized hypertrophied feeding cells necessary for growth and development of nematodes. The knot formation occurs with multiple infections, which cause the deformation of roots and the appearance of a chain-of- beads. In addition, infected roots cause deterioration of the plant as well as hindering the minerals and water uptakes, resulting in extremely decline of the plant growth. In the most 4 A. Suwanngam Validation of LAMP assay for specific-detection of M.mali in Belgium severe cases, the root system is completely destroyed when population density increases, leading to the easily overthrow of the plants (EPPO, 2017). Early detection is one of a prerequisite supporting role for effective prevention, control and elimination of pests. For Meloidogyne species, the classical taxonomy depending on morphological characteristics and perennial pattern assay of second-stage juvenile (J2) and adult females are commonly used ( Jepson, 1983; Handoo et al, 2005; Eisenback, 2014) . Although, traditional morphological identification assays relied upon microscopic examination, which limits its routine uses because of requiring an extra performing time, high experience and specialized skills. Unfortunately, misidentification occurred quite often with Meloidogyne mali, especially with close related species including M. arenaria and M. aredenensis (Itoh et al., 1969), M. hapla (Greco, 1981), M. suginamensis (Toida, 1984), M. thamesi (Toida and Yaegashi, 1991) and M. ulmi (Ahmed, 2013). Isozyme electrophoresis is another technique for nematode identification. This detection technique can be performed with adult females and second- stage juveniles has limitations resulting in unreliable diagnosis ( Esbenshade & Triantaphyllou, 1990). Moreover, the main drawback of this technique is time consuming, requires, manual skills and extra working resources such as the roots with females, microscope, microscopy slide, lactic acid and glycerin. Alternatively, molecular biology techniques such as PCR (Polymerase chain reaction) and LAMP (loop-mediated isothermal amplification) is a currently accepted reliable nematology detection method. The most desirable traits to develop detection methods is a straightforward implementation, precision and cost-effectiveness. PCR diagnostics is a highly reliable, sensitive, specific amplification and short-duration technique, which can be applied for several successful nematode detection assays as M. incognita, M. arenaria, M. hapla (Dong et al, 2001; Adam et al, 2007; Ye et al, 2015). Although, an important limitation in various PCR methods was the detection limits that must be considered to avoid 5 A. Suwanngam Validation of LAMP assay for specific-detection of M.mali in Belgium unreliable results. Recently, a novel molecular technique called LAMP was developed. LAMP is an isothermal nucleic acid sequence-based amplification technique that can amplify the target gene at a constant temperature with rapidity results in less than thirty minutes, highly sensitivity and specificity without the need of sophisticated instruments (Wong et al, 2017). Additionally, this technique is