The impact of the genus Meloidogyne in agricultural areas reinforces the need for an accurate diagnosis at species level. The development of a rapid and reliable method to identify the species found in the field increase the possibility of nematode control. In the past, RKN were identified by laborious microscopic examination of morphological and biometric characters which rely on measurements and comparison of morphological structures. According to Hirschmann (1985), the most useful characters in the identification of Meloidogyne species were the perineal pattern and the morphology of the female stylet, as well as the head shape, the distance of dorsal esophageal gland orifice to base of stylet and the stylet shape of males. In juveniles due to the small size, head characters were less useful, but tail length and shape were considered good characters because showed low intra-specific variation. However, diagnosis based on morphology is not always easy even for qualified taxonomists due to the great inter and intra-specific morphological and physiological variability and to the frequent occurrence of more than one species in the same sample (Einsenback, 1985). These problems led researchers to search for other methodologies to confirm and complement nematode species identification.
No differences were found in enzyme profiles within the same species even when nematodes were maintained in different hosts and according to Dickson et al., (1971) and Hussey et al., (1972) these patterns could be a useful parameter to be used for the taxonomy of the genus Meloidogyne. Since then, several studies were conducted with several enzymes and showing that the esterase phenotype of a single or few Meloidogyne spp. females is a reliable character for species identification (Esbenshade & Triantaphyllou, 1985, 1990; Fargette, 1987; Pais & Abrantes, 1989). Malate dehydrogenase, superoxide dismutase and glutamate-oxaloacetate enzymes were also often included to confirm species identification. Meloidogyne incognita and M. hapla can be separated with malate dehydrogenase, due to the difficulty in resolving size variants (Esbenshade & Triantaphyllou, 1985). The rapid and efficient biochemical electrophoretic analysis of non-specific esterases remain the first step in
the RKN species identification process, being very useful in the detection of populations with more than one species that can be easily separated to obtain pure isolates (Carneiro et al., 1996; Carneiro et al., 2000, 2004a; Castro et al., 2003; Cofcewicz et al., 2004; Hernandez et al., 2004; Abrantes et al., 2008; Brito et al., 2008). However, the observation of intra-specific variability, similarity between species and discovery of new esterase patterns make necessary not only the use of more than one enzyme phenotype but also additional information on biology and ecology of the nematode samples to confirm the identification (Esbenshade & Triantaphyllou, 1985; Cenis et al., 1992; Blok & Powers, 2009).
With the expansion of DNA-based methodologies, these have been developed and have shown to be useful, not only for nematode identification but also to provide important data on phylogenetic analysis. Methods based on polymerase chain reaction (PCR) are independent of the environmental influence and the nematode’s life cycle stage and potentially discriminatory (Zijlstra et al., 2000). Random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism variation (RFLP) and sequence characterized amplified regions (SCAR) markers have been developed and different regions of the DNA, including ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA), have been used to identify the RKN isolates (Cenis et al., 1992; Powers & Harris, 1993; Zijlstra et al., 1995, 2000, 2004; Randig et al., 2002; Xu et al., 2004; Adam et al., 2007).
The PCR-RFLP of mtDNA, namely the region flanked by the COII gene and the large (16S) ribosomal gene, has proved extremely useful in studies of characterization and identification of RKN species. This region encompasses partial COII and 16S rRNA sequences, the complete tRNA-His sequence, highly conserved in M. arenaria, M. floridensis, M. incognita and M. javanica, and an AT-rich non-coding sequence with different size, result of deletions giving rise to unique products of amplification enabling the differentiation of Meloidogyne spp. (Powers & Harris, 1993; Hugall et al., 1994, 1997; Stanton et al., 1997; Jeyaprakash et al., 2006). The AT-rich region is completely absent on M. hapla, and 167 bp, 573 bp, 603 bp, 963 bp and 1,100 bp products of amplification were found in M. mayaguensis, M. arenaria, M. floridensis, M. incognita and M. javanica, respectively (Jeyaprakash et al., 2006). PCR amplification of the region between COII and 16S rRNA genes, and subsequent digestion of the amplified products with restriction endonucleases discriminated five Meloidogyne spp. The amplified products of M. incognita and M. javanica resulted in a fragment of ca.
1,700 bp; M. arenaria produced a fragment of ca. 1,100 bp; and M. chitwoodi and M. hapla a fragment of ca. 520 bp. The restriction patterns produced by enzymes HinfI and DraI discriminated species with similar amplified products (Powers & Harris, 1993). Sequences of this region were also used to distinguish M. mayaguensis that produced a unique size product of ca. 705 bp; and M. floridensis from M. arenaria, M. mayaguensis, M. incognita and M. javanica by the size of amplified PCR products and the restriction pattern produced by enzyme SspI (Blok et al., 2002; Brito et al., 2004, Xu et al., 2004; Jeyaprakash et al., 2006).
This technique has been used widely in phylogenetic analysis, nematode characterization and identification and in various surveys providing not only species discrimination, but also revealing intra-specific variation among isolates (Blok et al., 2002; Brito et al., 2004; Xu et al., 2004; Handoo et al., 2005; Powers et al., 2005; Tigano et al., 2005; Jeyaprakash et al., 2006; Skantar et al., 2008; Devran et al., 2009). For instance, Meloidogyne arenaria (ca. 1,300 and 1,700 bp) and M. incognita (ca. 1,500 and 1,700 bp) have been reported as having populations that produced two sizes of PCR products (Powers & Harris, 1993; Blok et al., 2002; Xu et al., 2004; Tigano et al., 2005; Jeyaprakash et al., 2006).