Biological material

The juvenile J₂ of Meloidogyne were obtained using the Baermann funnel technique (^{Agrios, 2005}) of tomato plant roots with gall formations (Solanum lycopersicum), a farm located in San Nicolás de los Garza Nuevo León, provided by the Department of Plant Pathology of the Faculty of Life Sciences at the Autonomous University of Nuevo León, and identified as belonging to the Meloidogyne genus by taxonomic keys for the identification of parasitic plant nematodes (^{Zuckerman et al., 1990}).

Identification of the nematode

The genomic DNA was isolated from 5000 nematodes (J₂) using lysis with detergents followed by an extraction with phenol-chloroform-amyl alcohol (^{Sambrook and Russell, 2001}). The specimens were collected through centrifugation at 16,000 g for 5 minutes and the pellet was resuspended in 200 µL of lysis buffer TSNT (2 % Triton X-100, 1% SDS, 100 mM NaCl, 10 mM, TrisHCl and 1mM EDTA, pH 8), 500 µL of saturated phenol with Tris-HCl and 100 µL of a mixture of chloroform: amyl alcohol (24:1). The suspension was mixed in vortex for 5 minutes and 200 µL of buffer TE 1X were added (10mM Tris-HCl and 1 mM EDTA, pH 8.0); the phases were separated by centrifugation at 16,000 g for 10 minutes, the DNA settled on the aqueous phase with pure ethanol. The DNA pellets were dried at room temperature for 10 minutes and were resuspended in 48 µL of TE buffer, 2 µL of 2 µg µL-1 RNase and quantified through agarose gel densitometry dyed with GelRed 1X (Biotium, Hayward, CA) solution, using the EDAS 290 system and the Kodak Digital Science 1D (Eastman Kodak Company, Rochester, New York) program.

Afragment of rDNAwas amplified by PCR using the ITS1 (5´-TGAACCGGGCAAAAGTCG-3´) initiator and the ITS2 (5´-TTAGTTTCTTTTCCTCCGCT-3´) initiator, directed at the regions 3´ and 5´ of the rDNA 18S and 28S, respectively (^{Stock et al., 2001}; ^{Meza-García et al., 2014}). PCR was carried out in a TC020-24 MultiGene Mini (Labnet International, Inc., Edison, NJ) thermal cycler, with a final volume of 25 µL containing 1X buffer GoTaq PC (Promega, Madison, WI), 200 ng of genomic DNA, 200 µM of each dNTPs, 0.5 µM of each initiator, sterilized water and 1 U of GoTaq DNA Polymerase (Promega, Madison, WI). An amplification program of 32 cycles was used: 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 75 s, with a first denaturing stage of 94 °C for 3 min and a final elongation stage of 72 °C for 5 min. A plasmid that contains a fragment of ITS regions of Heterorhabditis indica Poinar, Karunakar & David 1992, (pGEMITSLZO) was used as a positive control of the PCR and the components of the PCR without DNA were used as negative control. The products of the PCR were analyzed in an electrophoresis with agarose gel 2.5 % dyed with GelRed 1X solution, using one unit of horizontal electrophoresis MSMUNIDUO (Sigma-Aldrich Co., St. Louis, MO), 100 volts for 60 minutes, and the product of the amplification was cloned in the pGEM(r)-T (Promega) vector, following the instructions of the manufacturer. The mixture of the ligation was used to transform cells of Escherichia coli DH5α, the transformed cells were selected through a white-blue test in agar slates LB with ampicillin (100µg mL-1), 100 µL IPTG (isopropil-β-D-1-thiogalactopyranoside) (2 mg mL-1). The resulting cell colonies of the transformed cells were analyzed in order to detect the presence of plasmid DNA through the extraction by alkaline lysis with an agarose gel 0.8 % dyed with GelRed 1X solution. Furthermore, the correct insertion of the amplified product in the pGEM(r)-T vector was confirmed through PCR using the ITS1 and ITS2 initiators, as was previously described.

The six positive plasmids by PCR of different colonies of E. coli were sequenced using the T7 and SP6 (Promega) initiator and a genetic analyzer ABI Prism 310 (Applied Biosystems, Foster City, CA) in the Unit of Molecular Biology of the Institute of Cellular Physiology of the Autonomous University of Mexico (UNAM).

The T7 and SP6 sequences (12 sequences in total) were aligned using the Contig Assembly Program module of the BioEdit v7.0.8.0 (^{Hall, 1999}) program and the consensus sequence was compared to the nucleotide sequences in the databases using the nucleotide BLAST (BLASTn) tool of the National Center of Information about Biotechnology (NCBI). The genus and species of the organism with the nucleotide sequence most related to the consensus sequence was considered as the genus and species of the analyzed nematode.