DOI: 10.5061/dryad.v6wwpzgtg
title: Morphological and molecular characterization of tropical Meloidogyne species in Brazil

description: 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.
description: <strong>Morphological
 identification</strong><br> 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. <strong>Genomic
 DNA extraction and common PCR from infective
 juveniles</strong><br> 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. <strong>Multiplex PCR and primer
 validation and molecular identification</strong><br>
 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. <strong>Gel
 electrophoresis</strong><br> 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.
description: In PCR reactions it is necessary to work with good quality DNA.
 And to know the efficiency of each process in the analysis of DNA, such as
 the extraction, quantification and purification of DNA. It is necessary to
 extract the intact DNA in order not to break the bonds, since these are
 responsible for the genetic information. Quantification involves
 estimating the DNA concentration obtained, which depends on the type and
 quantity of sample available.<br> DNA quantification by
 spectrophotometry is able to determine the average concentrations of
 nucleic acids DNA or RNA present in a sample, as well as their purity. To
 accurately assess the quality of the sample, 260/280 or 260/230 indices
 should be analyzed in combination with overall spectral quality. Pure
 nucleic acids typically produce a ratio of 260/280 to approximately 1.8
 and a ratio of 260/280 to approximately 2.0 for DNA. The 260/230 degree of
 purity is a second measure of DNA purity with the values ​​of a pure
 nucleic acid generally in the range of 1.8-2.2.
 The columns H onwards do not contain information, although column
 headings are included.