Biological material

The isolated strain of Clavibacter michiganensis ssp. michiganensis (Cmm) of Saladette tomato plants from Zamora Michoacán was provided by the phyto-bacterology lab from the Graduate College Campus Montecillo Montecillo, State of Mexico. The Cmm strain was kept on YDC selective medium (yeast extract, dextrose, CaCO₃ and agar) and propagated in LB liquid medium (Luria-Bertani) incubated for 48 h at 28 °C. Lycopersicon esculentum Mill plants Saladette type with symptoms and asymptomatic to Cmm, were collected in an open field in the locality "La Rinconada" de Zamora, Michoacán during the spring-summer 2013. For transformation chemically competent cells of Escherichia coli were used (Invitrogen™) TOP 10 F'. Plasmid TOPO TA 4 (Invitrogen™) was used for cloning the PCR product. Restriction enzyme Eco RI was used to release the cloned PCR products.

DNA extraction

To obtain DNA from Cmm the boling method (^{Holmes and Quigley, 1981}) was used. 1.5 mL Eppendorf tubes were prepared, 100 mg of glass beads of 450-600 μm and 150 of TE μL (Tris base, 50 mM EDTA, 2 mM) were added. Then transferred 2-4 mg of bacteria from solid plates with metal handle, in both plant material with symptoms and asymptomatic were transferred from 80-100 mg to each tube, in all cases were agitated with a vortex for 2 min. The tubes were sealed with parafilm, boiled for 5 min at 100 °C and mixed by stirring for 1 min. To each tube were added 150 μL of a phenol: chloroform (1: 1, v: v) mixture, stirred with the aid of a vortex for 1 min and centrifuged at room temperature at 12 000 rpm for 5 min. The supernatant of each sample was transferred to a new sterile Eppendorf tube, adding 100 μL of chloroform, vortexed for 1 min and centrifuged as in the previous step. 1 μL of each supernatant was used as temperate for PCR reaction. As control, DNA extraction was performed by the Dellapota method (^{Dellaporta et al., 1983}) recommended for plants. DNA isolation was verified by electrophoresis in agarose gel at 1.2% stained with GelRed™ (Biotum).

Polymerase chain reaction (PCR)

Amplification of a fragment form pat-1 gene (serine protease) of Cmm was performed by PCR using the oligonucleotides: CMM5F and CMM6R (^{Dreier et al., 1995}; Table 1); and as temperate the DNA obtained by the boiling method (^{Holmes and Quigley, 1981}). The components of the reaction mixture were: 17.4 µL of water, 2.5 µL of reg taq 10 X, 1.5 µL of MgCl₂, 1 µL of sense and antisense oligonucleotides, 0.5 µL dNTPs, 0.1 µL Taq and 1.0 µL of temperate DNA for total reaction volume 25 µL . Thermocycler GeneAmp PCR System 2400 Perkin conditions were: Denaturation 94 °C, 3 min, plus 35 cycles with the following temperatures and times: 94 °C, 30 s; alignment 55 °C, 1 min; synthesis 72 °C, 1 min and finally 1 additional cycle at 72 °C, 5 min, to obtain 250 bp amplicons a temperature annealing of 62 °C, for 30 s and synthesis 72 °C, 30 s (Table 1) were used. Amplification of PCR products were verified by electrophoresis on agarose gel 1.2% stained with GelRed™ (Biotum), the molecular size marker GeneRuler 1 kb DNA Ladder (Life Technologies) was used and the image was recorded on a transilluminator Vilber Lourmat (Marne-La-Vallee Cedex, France).

Cloning, sequencing and comparison

The PCR product was isolated and cloned into the vector pCR4-TOPO^®TA, colonies for plasmid DNA extraction were selected and digestion with Eco RI enzyme to verify the presence of the insert. Plasmid DNA purification of 2 clones was performed using QIAprep Spin Miniprep Kit Qiagen columns to obtain DNA-in quantity and quality for sequencing. Thus, the A_260nm /A_{280 nm} index was determined using the spectrophotometer Thermo Scientific^TM NanoDrop 2000/200c, which was close to 2.0. The oligonucleotide sequence was determined using the ABI PRISM 310 automated sequencer (Perkin Elmer, Norwalk, CT, USA). Comparison with the database was performed using the BLASTX algorithm (^{Altschul et al., 1990}) from the National Center for Biotechnology Information (NCBI).

Oligonucleotide Design

For the design of the 2 nested oligonucleotides pairs, melting temperature (Tm) and amplicon size 250 bp from pat-1 gene (serine protease) of Clavibacter michiganensis ssp. michiganensis, the algorithm OligoPerfect^TM Designer from Invitrogen (Table 1) was used.

Isolation of DNA Cmm

For identification of the virulent strain of Cmm, DNA extraction by boiling (^{Holmes and Quigley, 1981}) and Dellaporta (^{Dellaporta et al., 1983}) methods were used. The result is shown in Figure 1, where a discrete and upper band in size to10 000 bp corresponding to the genomic DNA of Cmm can be observed. The DNA obtained by the boiling method showed significant contamination by mRNA (lines 1 and 2), which is absent in the isolated DNA samples by the Dellaporta method (lines 3 and 4), on the other hand, the A_260nm / A_{280 nm} index for isolated plant DNA by the boiling method was 1.71, while for DNA isolated by Dellaporta method (Dellaporta et al., 1989) was 1.93. Additionally, the DNA isolated by the boiling method showed a concentration of 0.595 μg μL^-, as for the obtained by the Dellaporta method was 0.117μg μL^- (Table 2), suggesting that samples obtained by both methods can be used as temperate for PCR reactions.

Figure 1. DNA extraction of Clavibacter michiganensis ssp michiganensis (Cmm). Lines: MT, size marker; 1 and 2, DNA obtained by the boiling method; 3 and 4, DNA obtained by the Dellaporta method. 

Amplification of fragment pat-1 gene of Cmm

To reduce the time of analysis were used for the PCR reaction as temperate DNA of Cmm obtained by the boiling method and published oligonucleotides CMM5F and CMM6R (^{Dreier et al., 1995}; Table 1). The products obtained were separated on agarose gel 1.2% stained with GelRed ™ (Figure 2) showing amplification of a greater band than 500 bp (Line 1), which may correspond to the fragment pat-1 gene (serine protease), which has a size of 614 bp (Dreier et al., 1995). As negative control of PCR reaction (line 2), it was used as the temperate DNA obtained by boiling from healthy tomato plant and CMM5F and CMM6R oligonucleotides.

Figure 2. PCR amplification of a fragment of the pat-1 gene Cmm. Lines: MT, size marker; 1) using amplified fragment as temperate DNA of Cmm; and 2) negative control using as temperate DNA of asymptomatic tomato plant. 

Identification of Clavibacter michiganensis ssp. michiganensis (Cmm)

To identify the pathogenic strain Cmm, the PCR fragment of approximately 600 bp was cloned into the pCR4-TOPO®TA vector and transformed into Escherichia coli cells chemically competent. Plasmid DNA was purified to obtain the sequence, which is shown in Figure 3 where points out the CMM5F oligonucleotide and complement oligonucleotide CMM6R, suggesting that a fragment of 608 bp from pat-1 gene (serine protease) of Cmm has been cloned. The sequence obtained was compared with the database from the National Center for Biotechnology Information (NCBI) using the BLASTX algorithm (^{Altschul et al., 1990}) that translates the sequence and compares it to the registered proteins, the result is shown in Table 3, where sequences of some proteins of the strains with the respective access numbers are shown: Clavibacter michiganensis ssp. michiganensis (WP_011931204.1), C. michiganensis ssp michiganensis NCPPB 382 (CAM98537.1), C. michiganensis ssp. michiganensis (AEM65933.1), C. michiganensis ssp. michiganensis (AEM65932.1), among others, whose E value was close to 0.0 with pat-1 gene (serine protease) from Clavibacter michiganensis ssp. michiganensis, confirming the identification of pathogenic Cmm.

Figure 3. Base sequence of 608 bp fragment from pat-1 gene (serine protease) of Cmm. The sequences of the oligonucleotides CMM5F and the second chain CMM6R are underlined. 

Nested oligonucleotide design for the production of 250bp amplicons by PCR

Based on the sequence obtained two pairs of nested oligonucleotides to produce 250 bp amplicons were designed with an annealing temperature (Tm of 62 °C) which were designated arbitrarily HCMA1S-HCMB1As (20-mer) and HCMC2S- HCMD2As (21-mer). DNA isolated by the boiling method from tomato plants with symptoms to pathogenic Cmm were employed as temperate for PCR amplification of a fragment of the pat-1 gene (serine protease). The result is shown in Figure 4, where the amplification of a specific band of 250 bp with both pairs of nested oligonucleotides (lines 1 and 2) is observed. As a positive control a PCR was performed using as temperate the DNA of tomato plants with Cmm symptoms and CMM5F and CMM6R oligonucleotides, which generated the expected 608 bp fragment. As negative control, were used as temperate the DNA isolated from asymptomatic tomato plants to Cmm and the pair of nested oligonucleotides HCMA1S-HCMB1As, allowing the development of a rapid and specific PCR procedure, combining DNA extraction by the boiling method (^{Holmes and Quigley, 1981}) and nested oligonucleotides with Tm of 62 °C to produce amplicons of 250 bp from pat-1 gene (serine protease) for the identification of pathogenic Cmm.

Figure 4. PCR amplification of a 250 bp fragment from pat-1 gene of Cmm from DNA of plants with symptoms. Lines: MT, size marker; 1) nested oligonucleotides HCMA1S and HCMB1As; 2) nested oligonucleotides HCMC2S and HCMD2As; 3) positive control, oligonucleotides CMM5F and CMM6R; and 4) negative control, DNA plant from asymptomatic tomato as temperate and nested oligonucleotides HCMA1S and HCMB1As.