Surecycler 8800 thermocycler

Total RNA was extracted from the treated tomato leaves using RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions and dissolved in diethyl pyrocarbonate-treated water. The extracted RNA was then incubated with DNase for 1 h at 37 °C and quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific, USA).
Reverse transcription reaction was performed in a reaction mixture (20 µL) containing 2.5 µL 10×-buffer with MgCl₂, 2.5 µL of dNTPs (10 mM), 1 µL oligo (dT) primer (10 pmol µL⁻¹), 3 µL RNA (30 ng) and 0.2 µL reverse transcriptase enzyme (M-MuLV Reverse Transcriptase, Biolabs, NewEngland) and 10.8 µL sterile water. The PCR was performed using a SureCycler 8800 thermocycler (Agilent Technologies, USA) at 42 °C for 2 h, then at 70 °C for 5 min and the cDNA was then stored at −20 °C until used.

Sequencing of the D1/D2 region of the 26S rRNA gene was performed for all the isolates clustered (39 in total) using the primers NL-1 (5′-GCA TAT CAA TAA GCG GAG GAA AAG-3′) and NL-4 (5′-GGT CCG TGT TTC AAG ACG G-3′), as described by Van Der Aa Kühle and Jespersen [19 (link)]. Shortly, the PCR reaction was conducted in a 50 μL volume mixture containing 25 μL of Taq DNA Polymerase 2× Master Mix RED (Ampliqon, Odense, Denmark), 5 μL Primer mix NL-1 and NL-4, 17 μL sterile Milli-Q water, and 3 μL of the total DNA from yeast. The PCR reaction was carried out in a SureCycler 8800 thermocycler (Agilent Technologies, Santa Clara, CA, USA), using the following program: initial denaturation for 5 min at 95 °C, followed by 30 cycles at 95 °C for 90 s, 53 °C for 30 s, and 72 °C for 90 s, and the final elongation step at 72 °C for 7 min. The DNA sequencing (using the same primers; NL1 and NL4) was performed by Macrogen (Amsterdam, The Netherlands). Sequences were manually corrected, assembled with CLC Genomics Workbench version 7.9.1 software (QIAGEN Digital Insights, Redwood City, CA, USA), and compared to the reported 26S rRNA gene sequences in GenBank using the Basic Local Alignment Search Tool (BLAST) algorithm. The nucleotide sequences have been deposited in GenBank under Accession Numbers OL744629–OL744667, as indicated in Table 3.

Database searching and sequence analysis were carried out with the bioinformatics tools antiSMASH (Blin et al., 2013; Weber et al., 2015) and BLAST (Altschul et al., 1997). DNA manipulations were performed according to standard procedures for E. coli (Green and Sambrook, 2012) and Streptomyces (Kieser et al., 2000). PCR amplifications were carried out with Herculase II Fusion DNA Polymerase (Agilent Technologies, Madrid, Spain) following an optimized standard PCR procedure on a SureCycler 8800 thermocycler (Agilent Technologies): initial denaturation at 99.9°C for 2 min, 30 cycles comprised of 99.9°C denaturation for 10 s, 65°C annealing for 20 s and 72°C elongation at 30 s per kb of DNA to be amplified, plus an extra final cycle of 72°C for 3 min. Products of the expected size were cloned into pCR‐Blunt for sequence verification. All oligonucleotides used in this work are shown in Tables S1–S3 (Supporting information). PCR products were subsequently cloned into appropriate vectors using the selected restriction sites incorporated in the oligonucleotides.