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Ciência Rural

On-line version ISSN 1678-4596

Cienc. Rural vol.48 no.2 Santa Maria  2018  Epub Feb 08, 2018 


Diagnostic methods for identification of root-knot nematodes species from Brazil

Métodos diagnósticos usados na identificação de espécies do nematoide das galhas do Brasil

Tiago Garcia da Cunha1 

Liliane Evangelista Visôtto2  * 

Everaldo Antônio Lopes1 

Claúdio Marcelo Gonçalves Oliveira3 

Pedro Ivo Vieira Good God1 

1Instituto de Ciências Agrárias, Universidade Federal de Viçosa (UFV), Campus Rio Paranaíba, Rio Paranaíba, MG, Brasil.

2Instituto de Ciências Biológicas e da Saúde, Universidade Federal de Viçosa (UFV), Campus Rio Paranaíba, 38810-000, Rio Paranaíba, MG, Brasil.

3Instituto Biológico, Campinas, SP, Brasil.


The accurate identification of root-knot nematode (RKN) species (Meloidogyne spp.) is essential for implementing management strategies. Methods based on the morphology of adults, isozymes phenotypes and DNA analysis can be used for the diagnosis of RKN. Traditionally, RKN species are identified by the analysis of the perineal patterns and esterase phenotypes. For both procedures, mature females are required. Over the last few decades, accurate and rapid molecular techniques have been validated for RKN diagnosis, including eggs, juveniles and adults as DNA sources. Here, we emphasized the methods used for diagnosis of RKN, including emerging molecular techniques, focusing on the major species reported in Brazil.

Key words: DNA; esterase; Meloidogyne; molecular biology; morphological pattern


A identificação acurada de espécies do nematoide das galhas (NG) (Meloidogyne spp.) é essencial para a implementação de estratégias de manejo. Métodos baseados na morfologia de adultos, fenótipos de isoenzimas e análise de DNA podem ser usados para a diagnose do NG. Tradicionalmente, as espécies de NG são identificadas pela análise do padrão perineal e fenótipos de esterase. Em ambos os procedimentos, fêmeas maduras são necessárias. Nas últimas décadas, técnicas moleculares acuradas e rápidas têm sido validadas para a diagnose de NG, incluindo ovos, juvenis e adultos como fontes de DNA. Aqui, nós destacamos os métodos usados para a diagnose de NG, incluindo técnicas moleculares emergentes, focando nas principais espécies encontradas no Brasil.

Palavras-chave: DNA; esterase; Meloidogyne; biologia molecular; padrão morfológico


The genus Meloidogyne comprises more than 100 species and its host range exceeds 3000 species of plants (HUNT & HANDOO, 2009). These nematodes are spread worldwide, with a high diversity of species in tropical and subtropical regions. They cause damage to cash and subsistence crops all over the globe (LOPES & FERRAZ, 2016). RKN are sedentary endoparasites that induce the formation of giant cells in the roots, from which the nematode feed to complete its life cycle (BALDACCI-CRESP et al., 2015).The availability of water and nutrients to the plant decreases while the giant cells are located close to the root systems xylem and phloem. As the giant cells are located close to the xylem and phloem, availability of water and nutrients to the plant decreases (SIDDIQUI et al., 2014). Morphological and biochemical alterations induced by nematode parasitism cause abnormal growth of plants, nutrient deficiency symptoms, roots with galls, forking and other deformations (MOENS et al., 2009).

In Brazil, the major species of the root-knot nematode (RKN) are Meloidogyne incognita, M. javanica, M. arenaria, M. hapla, M. exigua, M. paranaensis, M. enterolobii (=Meloidogyne mayaguensis) and M. ethiopica (CARNEIRO et al., 2016). Accurate identification of RKN species is essential for implementing management strategies. For instance, the choice of resistant cultivars or cover crops for nematode control depends on the information of which species are dominant in the field. The coffee (Coffea arabica) cultivar ‘IAPAR 59’ is resistant to M. exigua and susceptible to M. paranaensis and M. incognita. Velvet bean (Mucuna pruriens var. utilis) does not allow M. incognita reproduction, but the same behavior is not observed when the nematode is M. javanica (FERRAZ et al., 2010).

For decades, observation of female perineal patterns was the routine method for RKN identification. Currently, isozyme phenotypes are used for diagnostics of RKN in many laboratories worldwide. However, in the last decade, several accurate and rapid molecular protocols for RKN diagnostics have been developed (SEESAO et al., 2017). Here, we discussed the morphological, biochemical and molecular methods used for the diagnosis of RKN, focusing on the major species reported in Brazil and including pros and cons of these techniques.

Diagnostic methods

Method based on morphology

Morphological characteristics of males

Head shape and stylet morphology of males are useful characteristics in the identification of some RKN species, such as M. incognita, M. enterolobii, M. paranaensis and M. javanica (Figure 1). For instance, male M. paranaensis can be identified based on its distinctive head morphology (Figure 1). For diagnostic purposes, specimens must be viewed in the lateral position. The distance from the dorsal esophageal gland orifice (DGO) to the stylet base of the males may also be used for the distinction between some species, such as M. enterolobii and M. incognita (ALMEIDA et al., 2008). The size and shape of the stylet also have a complementary taxonomical value for the identification of RKN species; although some RKN species share similarly sized stylets.

Figure 1 Anterior region of males of Meloidogyne incognita, M. enterolobii, M. paranaensis and M. javanica. DGO* = Dorsal esophageal gland orifice. 

Perineal pattern of females

Traditionally, the perineal pattern of females has been the main technique for RKN species identification. The shape and visual aspect of the whole perineal region, dorsal arch, dorsal striae, lateral lines and phasmids are the morphological characteristics used in identification. This is an inexpensive technique as it only requires a microscope, microscopy slide, coverslip, lactic acid and glycerin, but requires manual skills and it is time consuming during slide preparation. Also mature females are required, which means that roots with females must be available for diagnosis (SEESSAO et al., 2016). For images of perineal pattern of several RKN species, see FERRIS (1999).

In the late 1940s, this method was proposed as a tool for the identification of M. incognita, M. javanica, M. arenaria and M. hapla (CHITWOOD, 1949). However, with the discovery of new species of the pathogen, this technique proved not to be accurate enough to distinguish some species. The misidentification of M. enterolobii and M. inornata as M. incognita is a notorious example of this situation, since the perineal patterns of these species are similar (CARNEIRO et al., 2016). Such as for the use of morphological characteristics of males, the diagnosis of RKN species using this technique demands specialized taxonomic knowledge (OLIVEIRA et al., 2011).

Biochemical method

Isozyme phenotypes

Isoenzyme phenotyping is the routine diagnostic test for RKN in many laboratories worldwide. It is based on the relative mobility of enzymes extracted from mature females on gel electrophoresis (BLOK & POWERS, 2009). The whole procedure takes three to four hours, from sample processing to gel revelation. Protein extract from M. javanica females is applied on the gel for use as reference phenotype. Details on the method can be found in CARNEIRO & ALMEIDA (2001) and FREITAS et al. (2016).

Esterase phenotype (EST) analysis is usually enough to identify Meloidogyne species (ESBENSHADE & TRIANTAPHYLLOU, 1990; CARNEIRO et al., 1996; CARNEIRO & ALMEIDA, 2001) (Figure 2); although, other enzymes can also provide complementary information, such as malate dehydrogenase (MDH), superoxide dismutase (SOD) and glutamate oxaloacetate transaminase (GOT) (FREITAS et al., 2016). In some cases, EST are similar between two species, such as M. naasi and M. exigua. In this situation, MDH can be used to differentiate species (CARNEIRO et al., 2016).

Figure 2 Phenotypic profile of the esterase enzyme of the main Meloidogyne species reported in Brazil. Colorless boxes represent weak bands, filled boxes represent strong bands. Rm: Migration ratio in relation to the slower band (Rm = 1.0) of M. javanica. E1, I1, I2, J3, A2, H1, P1, Co2, En2 and E3: codes used to identify the esterase bands pattern of M. exigua, M. incognita, M. incognita = M. polycephannulata, M. javanica, M. arenaria, M. hapla, M. paranaensis, M. coffeicola, M. enterolobii and M. ethiopica, respectively. Source: Adapted from CARNEIRO et al. (2016). 

Intraspecific variations may occur in M. javanica, M. arenaria, M. exigua and M. paranaensis and other species, limiting the accuracy of the diagnosis (KUNIEDA et al., 1995; CARNEIRO et al., 1996; CARNEIRO et al., 2004; SALGADO et al., 2015). In addition, the procedure required the use of mature females and several individuals are needed in the case of species with small specimens, such as M. exigua (CARNEIRO et al., 2016).

Molecular methods

Molecular techniques rely on the occurrence of polymorphisms in DNA sequences among groups of nematodes, especially in nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA). Diagrams of rDNA and mtDNA of some RKN species can be found in VERONICO et al. (2004) and GARCÍA & SÁNCHEZ-PUERTA (2015). For identification and phylogenetic analysis of plant-parasitic nematodes, the genes 18S, 28S, 5.8S and the spacer regions (internal transcribed spacer - ITS, external transcribed spacer - ETS and intergenic spacer- IGS) have been the most studied rDNA regions, while the gene cytochrome c oxidase subunits I (COI, CO1 or COX 1) and II (COII, COII or COX 2) have been the main targets of mtDNA (ROBERTS et al., 2016).

The multicopy base of the rDNA contains variation and stability for discrimination of the major RKN species (ROBERTS et al., 2016). The small subunit of ribosomal DNA (18S rDNA or SSU) has been used in studies on inter-genera relationships, due to its low evolution rate (ROBERTS et al., 2016). Using a SSU as marker, 12 RKN species were grouped in three clades: I - M. incognita, M. arenaria and M. javanica; II - M. hapla races A and B, M. duytsi and M. maritima; III - M. exigua, M. graminicola and M. chitwoodi (DE LEY et al., 2002). The large subunit rDNA (28S rDNA or LSU) has conserved domains and expansion segments, such as the D2-D3 expansion region. This region is very useful for designing diagnostics for identification of species (ROBERTS et al., 2016). Several molecular protocols for nematode identification use spacer regions (ITS, ETS and IGS) as targets, especially ITS (ZIJLSTRA et al., 1995; BLOK et al., 1997; SCHMITZ et al., 1998; ORUI, 1999; ONKENDI & MOLELEKI, 2013). However, the presence of multiple ITS types within species or even within individuals may limit the accuracy of diagnostics that use ITS as a target (ROBERTS et al., 2016). The IGS region can be used to distinguish M. chitwoodi and M. fallax from other species, including M. enterolobii, M. hapla, M. incognita, M. javanica and M. arenaria (WISHART et al., 2002).

The mtDNA genes can be used in phylogenetic studies and for identification of RKN (KIEWNICK et al., 2014). Multiple copies of mtDNA are found per cell, which facilitates its amplification. In addition, it is highly conserved among animals and has variable regions flanked by conserved domains (ROBERTS et al., 2016). Despite these advantages as a marker, mtDNA has not been as studied as rDNA for the diagnosis of RKN.

Juveniles, eggs and adults can be used as DNA source. This is a great advantage of molecular methods in comparison to isozymes phenotypes and perineal pattern analysis, where mature females are required (OLIVEIRA et al., 2011). Furthermore, procedures used in molecular methods of nematodes are like those applied to other organisms, and do not require advanced knowledge on taxonomy of nematodes (BERRY et al., 2008).

The main molecular methods for the diagnosis of Meloidogyne species rely on the polymerase chain reaction (PCR), such as species-specific PCR, multiplex PCR, real-time PCR (qPCR) and RFLP (OLIVEIRA et al., 2011). Furthermore, isothermal amplification of loop-mediated DNA (LAMP), sequencing and DNA microarrays are useful for molecular diagnosis of RKN.

Species-specific PCR

In the last few decades, several species-specific primer sets have been developed for RKN diagnosis (Table 1). Primer design is crucial for the success of this approach. Species-specific primers must cover any intra-specific variation and not to amplify non-target nematodes (ROBERTS et al., 2016). Several primers designed for identification of tropical RKN species are based on SCAR (Sequence-characterized amplified region), including M. arenaria, M. incognita, M. javanica, M. paranaensis, M. exigua and M. enterolobii (Table 1). SCAR markers are developed from the characterization and sequencing of polymorphic bands resulting from RAPD (Random Amplified Polymorphic DNA) analysis (CARNEIRO et al., 2016).

Table 1 Universal and species-specific primers used in the identification of some Meloidogyne species through the polymerase chain reaction. 

Target Code Sequence (5’→3’) Amplicon Size (bp) Citation
Universal (5S - 18S rDNA) 194 TTAACTTGCCAGATCGGACG 720 BLOK et al. (1997)
Universal (D2/D3 28S rDNA) D2 ACAAGTACCGTGAGGGAAAGT 500 NUNN (1992)
M. arenaria (SCAR) - TCGAGGGCATCTAATAAAGG 950 DONG et al. (2001)
M. enterolobii (SCAR) - GAAATTGCTTTATTGTTACTAAG 322 BLOK et al. (2002)
M. enterolobii (SCAR) MK7-F GATCAGAGGCGGGCGCATTGCGA 520 TIGANO et al. (2010)
M. enterolobii (SCAR) Me-F AACTTTTGTGAAAGTGCCGCTG 236 LONG et al. (2006)
M. ethiopica (SCAR) meth-F ATGCAGCCGCAGGGAACGTAGTTG 350 CORREA et al. (2014)
M. exígua (SCAR) exD15-F CATCCGTGCTGTAGCTGCGAG 562 RANDIG et al. (2002)
M. hapla (SCAR) - GGCTGAGCATAGTAGATGATGTT 1500 DONG et al. (2001)
M. incognita (SCAR) - TAGGCAGTAGGTTGTCGGG 1350 DONG et al. (2001)
M. incognita (SCAR) Inc-K14-F GGGATGTGTAAATGCTCCTG 399 RANDIG et al. (2002)
M. incognita (SCAR) MI-F GTGAGGATTCAGCTCCCCAG 955 MENG et al. (2004)
M. incognita (SCAR) Mi2F4 ATGAAGCTAAGACTTTGGGCT 300 KIEWNICK et al. (2013)
M. javanica (SCAR) - CCTTAATGTCAACACTAGAGCC 1650 DONG et al. (2001)
M. javanica (SCAR) - ACGCTAGAATTCGACCCTGG 517 MENG et al. (2004)
M. paranaensis (SCAR) Par-F GCCCGACTCCATTTGACGGA 208 RANDIG et al. (2002)
M. hapla, M. chitwoodie M. fallax (IGS - SCAR) JMV2 TTTCCCCTTATGATGTTTACCC 440, 540, 670 WISHART et al. (2002)

Multiplex PCR

Two or more primers sets for different targets can be included in the same PCR reaction. As a result, amplification products of the distinct targets are visualized in a single gel, decreasing time, labor and costs. However, this approach requires similar reaction conditions for all the primer sets. The ability to detect species with low population densities in the sample may also be inhibited when a high concentration of DNA from another target is present. In a multiplex reaction, the DNA of Xiphinema elongatum amplified better than M. javanica DNA and, the latter, amplifies better than Pratylenchus zeae (BERRY et al., 2008).

Some protocols for multiplex PCR have already been developed for identification of RKN. Meloidogyne incognita, M. enterolobii and M. javanica, for example, can be identified by using three pairs of specific primers and one universal (HU et al, 2011). In this case, all species produce bands of approximately 500bp when universal primers (D2/D3) were used, while specific primers for M. incognita, M. enterolobii and M. javanica produced bands of approximately 1000, 200 and 700bp, respectively (HU et al., 2011). A multiplex assay can also identify tropical species M. incognita, M. javanica and M. arenaria (KIEWNICK et al., 2013).

Real-Time PCR (qPCR)

Conventional PCR is a qualitative method. A variation of this technique, real-time PCR or quantitative PCR (qPCR), allows the identification and real-time quantification of target sequences. qPCR is faster and more sensitive than conventional PCR, quantitative and does not require the preparation of gels. However, the high costs of equipment and reagents are still the main disadvantages of qPCR.

Real-time PCR detects and quantifies DNA based on the emission of fluorescence. The optical unit of the thermocycler monitors fluorescence emitted during cycling and the data is processed by a computer. The cycle threshold, that is, the number of cycles required to initiate the amplification of the target sequence, is then calculated. The two main systems used in the production of fluorescence in qPCR are hydrolysis probes (formerly Taqman®) and SYBR Green® dye. So far, qPCR-based diagnostics are available for M. arenaria, M. incognita, M. hapla, M. enterolobii and M. minor detection (Table 2). Despite its importance, no qPCR diagnostics is available for M. javanica.

Table 2 Primers for the identification of Meloidogyne species by real-time PCR (qPCR) and loop-mediated isothermal amplification (LAMP), including probes for qPCR. 

Target Code Sequence (5’→3’) Citation

*SYBR Green system. +Quarantine pest for Brazil. RKN: Universal sets for root-knot nematodes. Targets: Ma - M. arenaria; Me - M. enterolobii; Mi - M. incognita; Mh - M. hapla; Mm - M. minor. Citations: 1 - AGUDELO et al. (2011); 2 - TOYOTA et al. (2008); 3 - SAPKOTA et al. (2016); 4 - KIEWNICK et al. (2015); 5 - DE WEERDT et al. (2011); 6 - BERRY et al. (2008); 7 - NIU et al. (2011); 8 - NIU et al. (2012); 9 - PENG et al. (2017).

Restriction fragment length polymorphism (RFLP)

In this method, DNA is amplified by PCR using universal primers and then the products are digested by restriction endonuclease. As nucleotide sequences vary among species, restriction sites differ in their locations along the genome, resulting in fragments of different sizes. Finally, restriction products are separated by gel electrophoresis (SEESSAO et al., 2017). This technique allows the identification of M. hapla, M. incognita and M. arenaria (HAN et al., 2004). First, the PCR reaction is carried out by using primers that amplify the region between COII and LrRNA of mitochondrial DNA. Meloidogyne hapla sample will result in a 500bp band, while a 1.7 kb band is formed for M. incognita and M. arenaria DNA (HAN et al., 2004). To distinguish between the latter two species, the PCR products are cleaved with the restriction endonucleases Hinf I and Alu I, resulting in fragments of different sizes between M. incognita and M. arenaria, allowing their identification (HAN et al., 2004). The possible occurrence of false positives from non-target organisms is a disadvantage associated with PCR-RFLP (ROBERTS et al., 2016).

Loop-mediated isothermal amplification (LAMP)

The loop-mediated DNA amplification technique (LAMP) amplifies DNA with high specificity, sensitivity (up to 10 times more sensitive than PCR), efficiency and speed under isothermal conditions (NOTOMI et al., 2000). This procedure requires the use of four to six primers (FIP, BIP, F3, B3, with the possibility of one or two additional primers to increase the amplification efficiency - LB and LF) and a DNA polymerase (Bst polymerase) with strand displacement activity. The whole procedure takes from 30 minutes to less than 2 hours, depending on the protocol, and isothermal conditions (57 - 65ºC) can be reached by using simple equipment, such as water baths or heating blocks (NOTOMI et al., 2000). Amplification can be detected through visualization with naked eye, due to the formation of a white precipitate of magnesium pyrophosphate or the change of color of the solution by using dyes (SYBR Green, calcein, HNB, picogreen). This technique has been widely studied in many fields of science; although, few LAMP-based diagnostics for RKN have been developed so far (Table 2).


The availability of complete or partial genome sequences of nematodes is crucial to the development of molecular diagnostic protocols. Partial genome sequences from several Meloidogyne species have been published in open access databases, such as GenBank (<>). Complete genomes of M. incognita (ABAD et al., 2008) and M. hapla (OPPERMAN et al., 2008) are also available to scientists. The advent of next generation sequencing (NGS) technologies has reduced cost-per-base and time to decode partial or entire genomes. The number of genomic data tends to grow rapidly, as the new sequencers (2nd, 3rd and 4th generations) are faster than Sanger sequencing.

DNA microarrays

In this technique, a probe (cDNA or oligonucleotide) is hybridized with a fluorophore-labeled target molecule, usually a cDNA, produced from the conversion of total RNA or mRNA extracted from the sample. Several targets can be detected simultaneously, without any problems related to the competition of distinct targets for amplification, reduced specificity, occurrence of false-positives and difficulty of separation of the products in the gel, as can occur with multiplex PCR (FRANÇOIS et al., 2006). Despite the limited number of studies on the use of DNA microarrays in plant nematology, this technique has proven to be useful for the identification of M. chitwoodi (FRANÇOIS et al., 2006) and M. hapla (VAN DOORN et al., 2007).


The classical taxonomy, based on morphological and morphometric studies, and biochemical methods have been widely used in the identification of RKN species. Due to the limited number of taxonomic features and the declining interest in classical taxonomy, there is an increasing effort towards the development of molecular-based diagnostics. In general, molecular approaches are accurate, rapid and do not require only females for diagnosis purposes, such as isozyme phenotypes and perineal patterns. It is hoped that molecular techniques will soon be used more regularly in laboratories worldwide. However, scientists should use the integrative taxonomy approach for accurate diagnosis of RKN species. Molecular, biochemical and morphological data should be combined to enhance the resolution and reliability of studies on phylogenetics and etiology. For practical purposes, the information gathered in this review are expected to be useful for scientists, practitioners, students and other professionals interested in diagnosing RKN. As a consequence of an accurate identification, control strategies can be recommended and the losses caused by RKN can be decreased.


E. A. Lopes thanks Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)for the productivity grant (304663/2014-0).


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Received: July 03, 2017; Accepted: November 16, 2017; Revised: December 29, 2017

E-mail: *Corresponding author

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