Root-knot Nematode Species Research Articles

Identification and distribution of root-knot nematode species (Meloidogyne spp.) in vegetable growing areas of Lakes Region in Turkey

Calismada, Goller Bolgesi’nde yogun sebze uretimi yapilan alanlarda, Eylul 2014 – Aralik 2015 yillari arasinda toplanan kok-ur nematodu turleri morfolojik, molekuler ve Kuzey Karolina Konukcu Testi yontemleri kullanilarak karakterize edilmis ve yayilislari belirlenmistir. Toplam 160 adet ornek alinmis ve 83 tanesinin (%51.8) kok-ur nematodu ile bulasik oldugu bulunmustur. Urlu kok orneklerinden tek yumurta paketi alinarak duyarli ‘Tueza F1’ domates cesidinde her populasyonun saf kulturleri olusturulmus ve kitle uretimleri yapilmistir. Altmis sekiz populasyonun tur tanimlamalari morfolojik olarak disi bireylerin perineal bolge modelleri ve ikinci donem larva olcumlerinden ve molekuler olarak ture ozgu spesifik primerler kullanilarak yapilmistir. Tanimlanan 68 kok-ur nematodu populasyonundan 25 adedi M. incognita , 22 adedi M. hapla , 18 adedi M. javanica ve 1 adedi de M. arenaria olarak tespit edilmis, iki populasyonun ise tanilamalari yapilamamistir. Turlerin yayginlik oranlari sirasiyla, %36.7, %32.3, %26.5 ve %1.5 olarak belirlenmistir. Konukcu testine gore, M. incognita ’nin irk 2, irk 4 ve irk 6, M. javanica ’nin ise irk 1 ve irk 3’u belirlenmis ve M. javanica irk 3 Turkiye’de ilk kez rapor edilmistir. Meloidogyne incognita populasyonlarinin %84’u 800 m yukseltiye sahip mikroklima bolgelerde bulunurken, %16’si 800-1035 m yukseltiye sahip ortu alti alanlarda tespit edilmistir. Bazi M. javanica populasyonlari (%17) yuksek yayla bolgelerinde tespit edilirken, cogu (%84) alcak yukseltiye sahip sebze alanlarinda bulunmustur. Buna karsilik M. hapla populasyonlarinin buyuk cogunlugu (%91) daha yuksek serin bolgelerde ve kumsal toprak yapisina sahip alanlarda bulunurken, sadece %9 ‘u alcak yukseltiye sahip bolgelerde bulunmustur.

Identification of Two Meloidogyne hapla Genes and an Investigation of Their Roles in the Plant-Nematode Interaction.

Root-knot nematodes are soil-borne pathogens that invade and establish feeding sites in plant roots. They have an extremely broad host range, including most vascular plants. During infection of a susceptible host, root-knot nematodes secrete molecules called effectors that help them establish an intimate interaction with the plant and, at the same time, allow them to evade or suppress plant immune responses. Despite the fact that Meloidogyne hapla is a significant pest on several food crops, no effectors have been characterized from this root-knot nematode species thus far. Using the published genome and proteome from M. hapla, we have identified and characterized two genes, MhTTL2 and Mh265. MhTTL2 encodes a predicted secreted protein containing a transthyretin-like protein domain. The expression of MhTTL2 was up-regulated during parasitic life stages of the nematode, and in situ hybridization showed that MhTTL2 was expressed in the amphids, suggesting it has a role in the nematode nervous system during parasitism. We also studied the gene Mh265. The Mh265 transcript was localized to the subventral esophageal glands. An upregulation in Mh265 expression coincided with the pre- and early-parasitic life stages of the nematode. When Mh265 was constitutively expressed in plants, it enhanced their susceptibility to nematodes. These transgenic plants were also compromised in flg22-induced callose deposition, suggesting the Mh265 is modulating plant basal immune responses.

Soybean cyst nematode culture collections and field populations from North Carolina and Missouri reveal high incidences of infection by viruses.

Five viruses were previously discovered infecting soybean cyst nematodes (SCN; Heterodera glycines) from greenhouse cultures maintained in Illinois. In this study, the five viruses [ScNV, ScPV, ScRV, ScTV, and SbCNV-5] were detected within SCN greenhouse and field populations from North Carolina (NC) and Missouri (MO). The prevalence and titers of viruses in SCN from 43 greenhouse cultures and 25 field populations were analyzed using qRT-PCR. Viral titers within SCN greenhouse cultures were similar throughout juvenile development, and the presence of viral anti-genomic RNAs within egg, second-stage juvenile (J2), and pooled J3 and J4 stages suggests active viral replication within the nematode. Viruses were found at similar or lower levels within field populations of SCN compared with greenhouse cultures of North Carolina populations. Five greenhouse cultures harbored all five known viruses whereas in most populations a mixture of fewer viruses was detected. In contrast, three greenhouse cultures of similar descent to one another did not possess any detectable viruses and primarily differed in location of the cultures (NC versus MO). Several of these SCN viruses were also detected in Heterodera trifolii (clover cyst) and Heterodera schachtii (beet cyst), but not the other cyst, root-knot, or reniform nematode species tested. Viruses were not detected within soybean host plant tissue. If nematode infection with viruses is truly more common than first considered, the potential influence on nematode biology, pathogenicity, ecology, and control warrants continued investigation.

Diversity of Root-knot Nematodes Associated with Tubers of Yam (Dioscorea spp.) Established Using Isozyme Analysis and Mitochondrial DNA-based Identification.

The root-knot nematodes (RKN), Meloidogyne spp., represent an important threat to yam (Dioscorea spp.) production in West Africa. With the aim to establish the diversity of RKN species affecting yam tubers, for control and resistance screening purposes, surveys were conducted in the main yam producing areas of Nigeria. Galled tubers (N = 48) were collected from farmers' stores and markets in nine states in Nigeria and in one district in Ghana. RKN isolated from yam tubers were identified using enzyme phenotyping (esterase and malate dehydrogenase) and mitochondrial DNA (mtDNA) NADH dehydrogenase subunit 5 (Nad5) barcoding. Examination of 48 populations revealed that yam tubers were infested by Meloidogyne incognita (69%), followed by M. javanica (13%), M. enterolobii (2%), and M. arenaria (2%). Most of the tubers sampled (86%) were infected by a single species, and multiple species of RKN were detected in 14% of the samples. Results of both identification methods revealed the same species, confirming their accuracy for the identification of these tropical RKN species. In addition to M. incognita, M. javanica, and M. enterolobii, we report for the first time M. arenaria infecting yam tubers in Nigeria. This finding extends the list of yam pests and calls for caution when developing practices for yam pest management.

Identification of root knot nematode in thippali

Thippali, Piper longum, is an important medicinal plant belonging to the family Piperaceae. An increase in root knot nematode attack was observed in thippali growing plots in Kerala. But the nematode species has not been identified. The study was conducted to identify the root knot nematode attacking thippali at Department of Agricultural Entomology, College of Horticulture, Kerala Agricultural University, Vellanikkara, Thrissur, Kerala. The study was undertaken by collecting soil and root samples from thippali growing plots already infested with root knot nematode. The species of root knot nematodes were identified by the perineal pattern of white females collected from root galls. The white females collected from the infested roots were sent to Department of Nematology, Indian Agricultural Research Institute, New Delhi for species identification. The species of root knot nematode attacking thippali was identified as Meloidogyne arenaria.This is the first report on attack of M. arenaria on thippali from India. How to view point the article : Subhagan, Seena R. and Kurian, Susannamma (2016). Identification of root knot nematode in thippali. Internat. J. Plant Protec., 9(2) : 611-614, DOI : 10.15740/HAS/IJPP/9.2/611-614.

Meloidogyne species infesting tomatoes, cucumbers and eggplants grown in Kahramanmaraş Province, Turkey

In 2009 and 2010, 107 samples of roots and associated soil were collected from vegetable-growing fields in Andirin, Narli, Pazarcik and Turkoglu of Kahramanmaras Province, Turkey. Populations of root-knot nematode were found in tomatoes, eggplants and cucumbers and the species identified using isozymes analysis, mainly esterase resolved by PAGE (Polyacrylamide Gel Electrophoresis) and perineal patterns of nematode females. A single species of root-knot nematode, Meloidogyne incognita was identified from the samples collected. Two esterase phenotypes, one with a single band (I1) and the other with double bands (I2) were found at proportions of 8.5 and 15.8% of total samples, respectively. About 24% of the fields were found to be infested

English

In Uganda and elsewhere in the world, tomato is an economically important vegetable and a good source of vitamins A and C. Being an important horticultural crop, its production is threatened by root-knot nematode infections that make plants susceptible to wilting, growth reduction and infection by other pathogens like bacteria and fungi. Identifying the root-knot nematode species on the crop is paramount in designing proper management strategies especially crop rotation and resistance. In this study, tomato roots infected with Meloidogyne spp. were collected from fields in Kyenjojo and Masaka districts in Uganda. Using perineal patterns and molecular diagnostics, the three most common Meloidogyne species were identified. Meloidogyne javanica omit and was the most common followed by Meloidogyne arenaria and Meloidogyne incognita. Key words: Identification, root-knot nematodes, tomatoes, Uganda.

Distribution of root-knot nematode species and their virulence on vegetables in northern temperate agro-ecosystems of the Pakistani-administered territories of Azad Jammu and Kashmir

Root-knot nematodes (RKN) are yield devastating plant pathogens. Objective of this study was to investigate the distribution of RKN and their threat to vegetable production in the Pakistani-administered districts of Muzaffarabad, Hattian Bala and Neelum. A survey in more than 130 vegetable fields during summer 2012, 2013 and 2014 revealed a remarkable occurrence and prevalence of RKN in many vegetable crops. RKN were found in 64 % of the fields in Muzaffarabad, 30 % of the fields in Hattian Bala and 24 % of the fields in Neelum. Okra was infested on 26 sites with a gall index range of 3–10, tomato on 26 sites with a gall index of 2–10, cucumber at 13 sites with a gall index of 3–9 and eggplant at 11 sites with a gall index of 3–9. Other hosts infested by RKN included mint, beans, cucurbits and peach. Identification of RKN species was performed morphologically by perineal patterns, by PCR using primers for the IGS2 region or SCAR primers, and some by sequencing of a COI gene fragment. The RKN of this temperate region were mostly identified as tropical species. M. incognita was found in 83 % of the tested fields followed by M. javanica (76 %) and M. arenaria (36 %). M. javanica was the predominant species in Muzaffarabad and Hattian Bala. This is the first comprehensive study on the presence of tropical RKN species in the temperate region of Azad Jammu and Kashmir, Pakistan, and their virulence on vegetable crops.

Grapevine rootstocks resistant to the root-knot nematodeMeloidogyne javanica

Background and Aims Root-knot nematodes (Meloidogyne spp.) are endo-parasites of plant roots and parasitisation can lead to diminished grape yields. Worldwide viticulture production is impacted primarily by four species of root-knot nematode (Meloidogyne arenaria, Meloidogyne hapla, Meloidogyne incognita and Meloidogyne javanica). Meloidogyne javanica is the predominant root-knot nematode found in Australian vineyards. A glasshouse-based experiment was conducted to identify grapevine cultivars and accessions with complete resistance (i.e. no reproduction of nematode) to an aggressive pathotype of M. javanica isolated from an Australian vineyard. Methods and Results Single-grapevine plants were inoculated with approximately 1500 second stage juveniles of M. javanica in a replicated study. Six weeks after inoculation, roots were washed free of soil, and egg masses were stained and tallied. The final dry mass of roots was measured to determine the ratio of egg masses to root mass. Conclusions Complete resistance to M. javanica was found in 42 of 75 Vitis cultivars and accessions screened. Significance of Study The M. javanica resistant cultivars and accessions identified in this study provide valuable material for future breeding in order to develop new rootstocks for the Australian wine and grape industry with durable resistance to root-knot nematode.

Morphological, Biometrical, Biochemical, and Molecular Characterization of the Coffee Root-Knot Nematode Meloidogyne megadora

Meloidogyne megadora infects coffee trees, an economically important crop worldwide. The accurate identification of M. megadora is essential for the development of preventive measures to avoid the dispersion of this pathogen and establishment of efficient and sustainable integrated pest management programs. One M. megadora isolate was studied by biometrical, biochemical, and molecular characteristics (random amplified polymorphic DNA [RAPD] and PCR of internal transcribed spacer [ITS] region). Biometrical characteristics of M. megadora females, males, and second-stage juveniles were similar to the original description. Biochemical studies revealed a unique enzyme pattern for M. megadora esterases (Me3) that allowed for species differentiation. Three RAPD primers (OPG-4, OPG-5, and OPG-6) produced specific bands to all Meloidogyne spp. studied: M. megadora, M. arenaria, M. incognita, and M. javanica. Molecular analysis of the ITS region resulted in an amplification product of 700 bp. The phylogenetic relationship between M. megadora and several Meloidogyne spp. sequences was analyzed, revealing that M. megadora clearly differs from the most common root-knot nematode species. Based on the studies conducted, isozyme analysis remains a useful and efficient methodology for M. megadora identification when females are available. Further studies will be needed to convert the M. megadora differential DNA fragment obtained by RAPD and develop a species-specific sequence-characterized amplified region PCR assay for its diagnosis based on second-stage juveniles.

Plant Genetic Background Increasing the Efficiency and Durability of Major Resistance Genes to Root-knot Nematodes Can Be Resolved into a Few Resistance QTLs.

With the banning of most chemical nematicides, the control of root-knot nematodes (RKNs) in vegetable crops is now based essentially on the deployment of single, major resistance genes (R-genes). However, these genes are rare and their efficacy is threatened by the capacity of RKNs to adapt. In pepper, several dominant R-genes are effective against RKNs, and their efficacy and durability have been shown to be greater in a partially resistant genetic background. However, the genetic determinants of this partial resistance were unknown. Here, a quantitative trait loci (QTL) analysis was performed on the F2:3 population from the cross between Yolo Wonder, an accession considered partially resistant or resistant, depending on the RKN species, and Doux Long des Landes, a susceptible cultivar. A genetic linkage map was constructed from 130 F2 individuals, and the 130 F3 families were tested for resistance to the three main RKN species, Meloidogyne incognita, M. arenaria, and M. javanica. For the first time in the pepper-RKN pathosystem, four major QTLs were identified and mapped to two clusters. The cluster on chromosome P1 includes three tightly linked QTLs with specific effects against individual RKN species. The fourth QTL, providing specific resistance to M. javanica, mapped to pepper chromosome P9, which is known to carry multiple NBS–LRR repeats, together with major R-genes for resistance to nematodes and other pathogens. The newly discovered cluster on chromosome P1 has a broad spectrum of action with major additive effects on resistance. These data highlight the role of host QTLs involved in plant-RKN interactions and provide innovative potential for the breeding of new pepper cultivars or rootstocks combining quantitative resistance and major R-genes, to increase both the efficacy and durability of RKN control by resistance genes.

Screening of Tomato Genotypes Against Root-Knot Nematode and Validation of Mi 1 Gene Linked Markers

In India, tomato (Solanum lycopersicum L.) suffers yield loss between 11 and 35 % due to root-knot nematode (RKN) (Meloidogyne spp.) infestation. Mi 1 gene in tomato confers resistance to the three most damaging RKN species viz, Meloidogyne incognita, M. arenaria and M. javanica. The loss can be averted through the use of cultivars containing Mi 1 gene or incorporating the gene in new cultivars. To identify resistant genotypes, in 2013–2014 a total of 32 genotypes were screened against M. incognita at 2000 J2/Kg inoculum. Genotypes, Motelle and H-88-78-1 have showed immune reaction. Mogor and Hisar Lalit also showed resistance reaction. In 2014–2015, nine genotypes including four immune/resistant lines from the last year were screened against RKN at two inoculation rates of 2000 and 4000 J2. Reaction of Motelle was similar at both inoculum levels. In other resistant genotypes there was increase in mean of gall index when the inoculum levels were increased. The genetic background had an effect on the variations observed in number of galls produced in the resistant genotypes. Marker assisted selection for resistant genes greatly enhance selection of resistant plants in breeding. To identify suitable marker, six polymerase chain reaction based co-dominant markers were used for molecular screening of Mi 1 gene. Four markers namely, REX-1, CT119, Mi23 and Pmi gave banding pattern in accordance with the reported literature. Considering tight linkage with the gene and assay cost two sequences characterized amplified region markers, Mi23 and Pmi can be preferred over other markers for selecting Mi 1 containing genotypes.

First Report of Meloidogyne haplanaria Infecting Mi-Resistant Tomato Plants in Florida and Its Molecular Diagnosis Based on Mitochondrial Haplotype.

Meloidogyne haplanaria is a species originally found infesting peanut in Texas and, more recently, in Arkansas. In this study, we confirmed the presence of M. haplanaria in Florida based on morphological and molecular characterization. This species was identified from a sample submitted for diagnosis collected from Mi-resistant tomato rootstock grown in Naples, FL. The major diagnostic criteria to distinguish M. haplanaria from other closely related root-knot nematode (RKN) species are based on morphological differences and host range tests, which are time consuming and labor intensive and require living or well-preserved specimens. In our study, we provide an easy diagnostic strategy to distinguish M. haplanaria from other RKN species based on amplification of two mitochondrial DNA regions. These regions span the intergenic spacer and part of the adjacent large subunit ribosomal RNA gene (lrDNA) and sequence polymorphisms in lrDNA revealed by the restriction pattern following digestion with the restriction enzymes HinfI and MnlI. A unique haplotype pattern, which has not been observed in any of the RKN species described thus far, was observed in M. haplanaria. The outcome of molecular analysis of M. haplanaria aligned with morphological measurement and characteristics as well as perineal pattern originally described for M. haplanaria.

Survey and Identification of Root-Knot Nematodes Associated with Brinjal Crops in Fatehabad, Agra

A survey was conducted over a period of one and half year to estimate the occurrence of root-knot nematode disease on brinjal crops in five selected localities in and around Fatehabad, Agra. Our survey indicates that brinjal crops grown in the selected localities were heavily infested with root-knot nematodes. Highest frequency of disease occurrence in which almost all the roots have knot-like appearance (>85%) was reported from Firozabad Road area. Other localities were also having the significant infestations and these were reported as 80%, 78%, 65%,62% respectively. To access the damage caused by root-knot nematodes in brinjal crop, Meloidogyne gall index and its egg-mass index were calculated and these were found in the range of 2-5. An increased gall index and egg-mass index point to heavy infection on selected crops. Sampled crop showed the significant presence of Meloidogyne javanica. However, there were other species of root-knot nematode as mixed population was also reported. Other species of the root-knot nematodes which were reported in this survey was M. incognita. This survey indicates that the M. javanica is a frequently occurring population in brinjal field infested with root-knot disease.

Effect of selected planting dates on root- knot nematodes population and productivity of sugarbeet variety, Pleno

The effect of three planting dates (August 25, September and October) on infectivity of root- knot nematode species and productivity of sugar beet var. Pleno was evaluated. The results indicated that the 1st planting date showed the highest values of nematode infection parameters (i.e. numbers of galls, eggmasses, development stages root system-1, juveniles in soil and final population as well as reproduction factor) followed by 2nd planting date. However, the 3rd planting date achieved the lowest values of those nematode parameters. In addition, the data revealed that the 1st planting date obtained only the highest values for sucrose and purity percentages. On the other hand, the 3rd planting date recorded the highest values of roots, top, biological and sugar yields (tons fed-1). Therefore, the 3rd planting date (during October) was the most effective date against concerned species of root-knot nematodes on naturally infested sugar beet grown in sandy soil using drip irrigation system.

Identifikasi Spesies Meloidogyne spp. Penyebab Umbi Bercabang pada Tanaman Wortel di Jawa Timur

Branched tuber is a new disease in carrot cultivation in Indonesia and has caused big losses for farmers. In 2010, several species of root-knot nematodes, Meloidogyne spp ., were reported as the primary cause of disease and the yield loss due to the disease in the region of Agropolitan-Cianjur, West Java, reached 15% to 95%. Symptoms of tuber branching has also been reported in one of the centers of vegetable production in East Java, in the subdistrict of Bumiaji, Kota Batu. Research aimed at identifying Meloidogyne species on carrot was carried out on carrot plantation. The study consisted of two activities, i.e. surveillance and identification. Surveillance was carried out at three different altitudes/elevations, i.e. 1600 m, 1700 m and 1800 m asl (above sea level). Identification was done by PCR ITS r-DNA using multiplex primer for mix spesies identification of M. hapla, M. fallax, and M. chitwoodi and the spesific primer for identification of M. arenaria, M. incognita , and M. javanica . Identification based on ITS r-NA by PCR gave positive results for four species, namely M. hapla, M. arenaria, M. incognita , and M. javanica. Key words: branched tuber disease, carrot, Meloidogyne , PCR ITS r-DNA

IPR AFRODITE - new oat cultivar with nematode resistance

The oat cultivar IPR Afrodite, resulting from a cross between CFT 2 and the inbred line ER 88144-1, is high-yielding, has grain quality traits that are relevant for food processing and industry, and is resistant to the root-knot nematode species Meloidogyne incognita, M. javanica, M. paranaensis and M. enterolobii.

Nematode quantitative resistance conferred by the pepper genetic background presents additive effects and is stable against different isolates of Meloidogyne incognita

Root‐knot nematodes (RKNs), Meloidogyne spp., are a major disease problem in solanaceous crops worldwide, including pepper (Capsicum spp.). Genetic control provides an economically and environmentally sustainable protection alternative to soil fumigants. In pepper, resistance to the main RKN species (M. incognita, M. javanica and M. arenaria) is conferred by the major genes (R genes) Me1, Me3 and N. However, RKNs are able to develop virulence, thus endangering the efficiency of R genes. Quantitative resistance (QR) against Meloidogyne spp. is expected to provide an alternative to R genes, or to be combined with R genes, to increase the resistance efficiency and durability in pepper. In order to explore the ability of QR to protect pepper against RKNs, five pepper inbred lines, differing in their QR level, were tested directly, or after combination with the Me1 and Me3 genes, for their resistance to a panel of M. arenaria, M. javanica and M. incognita isolates. The M. arenaria and M. javanica isolates showed low pathogenicity to pepper, unlike the M. incognita isolates. The QR, controlled by the pepper genetic background, displayed a high resistance level with a broad spectrum of action, protecting pepper against Me3‐virulent as well as avirulent M. incognita isolates. The QR was also expressed when combined with the Me1 and Me3 genes, but presented additive genetic effects so that heterozygous F1 hybrids proved less resistant than homozygous inbred lines. The discovery of this QR is expected to provide promising applications for preserving the efficiency and durability of nematode resistance.

Parasitism effects on white clover by root-knot and cyst nematodes and molecular separation of Heterodera daverti from H. trifolii

This research carried out an accurate identification of the root-knot and cyst-forming nematode species parasitizing white clover at the Laceno Lake area in Southern Italy. Two species, the root-knot nematode Meloidogyne hapla and the cyst nematode Heterodera daverti were identified by integrative taxonomic approaches (classical, isozyme pattern, and D2-D3 expansion segments of 28S rRNA, ITS rRNA and coxI of mtDNA gene sequences) and found parasitizing white clover roots. These nematodes were detected in stunted plants with a reduced number of rhizobium nodules and the host suitability was confirmed by the high nematode population densities ranging from 53 to 2350 eggs and J2s per g of fresh roots for M. hapla, and 1.36 eggs and J2s/cm3 of soil for H. daverti; and cyst nematode females were also detected on the roots of clover. The studies on the host-parasite relationships of nematode-feeding sites in white clover roots infected by these nematodes showed a high susceptible response. Meloidogyne hapla and H. daverti infections were also observed on nitrogen-fixing root nodules of white clover, where well established feeding sites allowed active nematode reproduction. Histological examination of nitrogen-fixing root nodule tissues revealed that the nematodes established their permanent feeding sites in the vascular bundles of nodules which appeared enlarged deformed and disorganised by the expansion of nematode feeding cells (giant cells and syncytium) and hyperplasia of the nodule cortex. Additionally, coxI of mtDNA gene is an efficient barcoding sequence for discriminating the identification of H. daverti from H. trifolii.

Development and Validation of LNA-Based Quantitative Real-Time PCR Assays for Detection and Identification of the Root-Knot Nematode Meloidogyne enterolobii in Complex DNA Backgrounds.

Meloidogyne enterolobii is a quarantine root-knot nematode posing a major threat to agricultural production systems worldwide. It attacks many host plants, including important agricultural crops, ornamentals, and trees. M. enterolobii is a highly virulent and pathogenic root-knot nematode species, able to reproduce on plants resistant to other Meloidogyne spp. Significant crop damage has been reported in Asia, South America, Africa, the United States, France, and greenhouses in Switzerland. To identify potential introduction pathways and ensure appropriate phytosanitary measures and management strategies, accurate detection and identification tools are needed. Therefore, two real-time quantitative polymerase chain reaction (PCR) assays based on the second intergenic spacer region of the ribosomal DNA cistron and the cytochrome oxidase c subunit I (COI) gene using locked nucleic acid probes were developed and validated for fast and reliable detection and identification of M. enterolobii. Analytical specificity was confirmed with 16 M. enterolobii populations, 16 populations of eight closely related Meloidogyne spp., and four species from other nematode genera. Optimizing and testing the assays on two real-time PCR platforms revealed an analytical sensitivity of one juvenile in a background of 1,000 nematodes and the intended limit of detection of one juvenile per 100 ml of soil. Both assays performed equally well, with the COI-based assay showing a slightly better performance concerning detection of M. enterolobii target DNA in complex DNA backgrounds.