Rapid detection of Pecan Root-Knot Nematode,Meloidogyne partityla, in laboratory and field conditions using loop-mediated isothermal amplification

Abstract

2Meloidogyne partityla is the dominant root-knot nematode (RKN) species parasitizing pecan in 3 Georgia. This species is known to cause a reduction in root growth and a decline in yields from 4 mature pecan trees. Rapid and accurate diagnosis of this RKN is required to control this nematode 5 disease and reduce losses in pecan production. In this study, a loop-mediated isothermal 6 amplification (LAMP) method was developed for simple, rapid and on-site detection of M. 7 partityla in infested plant roots and validated to detect the nematode in laboratory and field 8 conditions. Specific primers were designed based on the sequence distinction of internal 9 transcribed spacer (ITS)-18S/5.8S ribosomal RNA gene between M. partityla and other 10 Meloidogyne spp. The LAMP detection technique could detect the presence of M. partityla 11 genomic DNA at a concentration as low as 1 pg, and no cross reactivity was found with DNA from 12 other major RKN species such as M. javanica, M. incognita and M. arenaria, and M. hapla. We 13 also conducted a traditional morphology-based diagnostic assay and conventional polymerase 14 chain reaction (PCR) assay to determine which of these techniques was less time consuming, more 15 sensitive, and convenient to use in the field. The LAMP assay provided more rapid results, 16 amplifying the target nematode species in less than 60 min at 65°C, with results 100 times more 17 sensitive than conventional PCR (~2-3 hrs). Morphology-based, traditional diagnosis was highly 18 time-consuming (2 days) and more laborious than conventional PCR and LAMP assays. These 19 features greatly simplified the operating procedure and made the assay a powerful tool for rapid, 20 on-site detection of pecan RKN, M. partityla. The LAMP assay will facilitate accurate pecan 21 nematode diagnosis in the field and contribute to the management of the pathogen. 3 1 diagnosis 3 Introduction 4 Pecan (Carya illinoinensis) is an important nut crop in North America. A variety of diseases caused 5 by bacteria, fungi, virus, and nematode can attack these trees, and if not properly managed, they 6 can cause economic damage to pecans. Root-knot nematodes (RKNs) of the genus Meloidogyne 7 are economically important plant-parasitic nematodes, which cause significant damage to pecan 8 production. Three species of RKNs, Meloidogyne incognita, M. arenaria and M. partityla have, 9 have been reported as pathogenic to pecan [1-3]. Among these species M. partityla is the dominant 10 RKN parasitizing pecan which has a greater incidence in southern United States [2-6]. M. partityla 11 was originally found infecting pecan in South Africa, and was likely introduced into the United 12 States during importation of infected pecan seedlings [7]. Due to the complexity and lengthy 13 process of diagnosis and species identification, it is difficult to conduct extensive surveys, which 14are required for determining the pecan RKN distribution. Therefore, a quick diagnosis approach 15 will be helpful to know the latest status of the pecan RKNs in s...