Morphological and molecular characterization of tropical Meloidogyne species in Brazil

The identification of Meloidogyne species, based on the perineal pattern of females and the morphology of the anterior portion of males, requires knowledge of the characteristics of each species and, mainly, training and practice. Molecular techniques, applying the concept of integrative taxonomy, have validated the diagnosis of plant-parasitic nematodes through DNA analysis. The objective of the present study was to evaluate the accuracy and feasibility of molecular techniques to identify and discriminate Meloidogyne species that are more common in tropical regions. This was done aiming to validate the multiplex PCR technique for the diagnosis of these organisms and to complement the identification based on morphology. Specific primers, used in molecular techniques, to identify the main Meloidogyne species in tropical regions and multiplex PCR, for multiple and rapid detection, were tested. The primers Fjav-1 and Rjav-1, Fexi-1 and Rexi-1, Finc-1 and Rinc-1, and Fpar-1 and Rpar-1, except Fent-1/Rent-1 for M. enterolobii and the conditions of PCR, are adequate to detect and discriminate M. enterolobii, M. exigua, M. incognita, M. javanica and M. paranaensis. This approach, which only requires the DNA extracted from the nematodes, is a simple, fast and reliable diagnostic technique for the main tropical Meloidogyne species.
Methods
Morphological identification

The morphological identification of  nematodes species, followed the criteria established in their original descriptions (Eisenback and Hirschmann, 1981). Ten fully developed and milky females per species were removed from the roots and stored in water for perineal cuts. Each female was transferred to a slide containing a drop of lactic acid (45%) under a stereomicroscope and a cross section was made in the anterior region of each one, with the aid of a scalpel, for the complete extravasation of their internal content. The perineal region was dissected and transferred to a slide, containing glycerin (100%), sealed and labeled (Hussey & Barker 1973). Specimens with predominant characteristics of the group were selected and used. The shape and appearance of the perineal region, dorsal arch, dorsal streaks and lines of the lateral fields of the females were the morphological characteristics observed. The nematode males were obtained from processed roots, whose suspension was observed under a stereomicroscope.Ten males were recovered from the suspension, by species, using a fishing rod and transferred to a temporary, sealed and labeled blade containing water (Hussey & Barker 1973). The identification of the nematode males was based on their anterior region. Morphological characteristics of females and males were analyzed using an Olympus “BX50” microscope with an attached Olympus “DP72” camera.

Genomic DNA extraction and common PCR from infective juveniles

The genomic DNA of 50 infective juveniles of four Meloidogyne species (Table 1) was extracted in 50 µl of 0.85% NaCl, by Worm Lysis Buffer (WLB)  (Carvalho et al., 2019). These samples were placed in solution with KCl (50 mM), Tris (10 mM pH 8.2), MgCl2 (2.5 mM), Tween 20 (0.45%) and proteinase K (200 mg/ml) at -70ºC for 15 minutes, incubated for one hour at 60ºC, 15 minutes at 95ºC and stored at -20ºC. The quality of the extracted DNA was verified by a universal primer D2a and D3b (Nadler et al., 1999) amplifying the D2 and D3 of the expansion region of the 28D rDNA nuclear gene. Positive samples, for the primers D2a and D3b, and the DNA of M. enterolobii, M. exigua, M. incognita, M. javanica and M. paranaensis were amplified with specific primers (Table 2). The amplification reactions were carried out with 12.5 μl of Dropsq (Neobio), 1 μl of each primer [10 p/mol] and 7.5 μl of nuclease-free water 3 μl of the DNA mixture totaling a 25 μl reaction.

Multiplex PCR and primer validation and molecular identification

Meloidogyne exigua, M. incognita, M. javanica and M. paranaensis were detected and identified, simultaneously, by PCR, each, quickly, with the four pairs of specific primers. The primers Fjav-1 and Rjav-1, Fexi-1 and Rexi-1, Finc-1 and Rinc-1 and Fpar-1 and Rpar-1 were used for the multiplex PCR for M. exigua, M. incognita, M. javanica, and M. paranaensis, respectively. The 500 bp fragment, resulting from universal primers, is an internal positive of DNAr 28S D2/D3.The amplification reactions were performed in 0.2 ml of PCR tubes with reaction volumes of 25 μl. The main mixture had 12.5 μl of drq (Neobio), 0.5 μl of each primer [10 w/mol], 5.5 μl of nuclease-free water and 3 μl of the DNA mixture. The DNA quality was verified with the fragment D2/D3 extension of the 28S rDNA to avoid false negatives in the specific PCRs by species of nematode.  The confidence of the nematode identification using the primers (Table 2), based on PCR  withthe the extraction of genomic DNA from 50 infective juveniles to verify amplification (Fig. 3) and specific ones was greater.

Gel electrophoresis

Five μl of PCR product from each sample was loaded onto the agarose gel. The DNA bands were stained with GelRed (Neobio) and visualized and photographed on a UV transilluminator (Major Science). The remaining products, per sample, were stored at -20°C.