Table 1 Molecular characterization of potential biocontrol isolates
(a) Base sequences of 16S rDNA and ITS rDNA primers used | |||
|---|---|---|---|
Primers | Primer ID | Sequence | Base pairs |
16S rDNA | 27F | 5′-AGAGTTTGATCCTGGCTCAG-3′ | 20 |
|  | 1492R | 5′-TACGGYTACCTTGTTACGACTT-3′ | 22 |
ITS rDNA | ITS1F | 5′-TCCGTAGGTGAACCTGCGG-3′ | 19 |
|  | ITS4 | 5′-TCCTCCGCTTATTGATATGC-3′ | 20 |
(b) PCR mixtures for 10 μl and 50 μl reaction volumes | |||
|---|---|---|---|
Components | Quantity for one reaction | ||
Total volume (10 μl) | Total volume (50 μl) | ||
EmeraldAmp GT PCR Master Mix (2X premix) | 5 μl | 25 μl | |
Primers (2.5 pmol/μl) | |||
 Forward | 1 μl | 5 μl | |
 Reverse | 1 μl | 5 μl | |
Template DNA (100 ng/μl) | 1 μl | 5 μl | |
dH2O | 2 μl | 10 μl | |
Total volume | 10 μl | 50 µl | |
(c) Cycling conditions and amplicon size for 16S rDNA and ITS rDNA amplification | |||
|---|---|---|---|
Step | 16S rDNA | ITS rDNA | |
Initial denaturation | 96 °C for 4 min | 94 °C for 5 min | |
35 cycles of | |||
 Final denaturation | 94 °C for 40 s | 94 °C for 45 s | |
 Primer annealing | 57 °C for 1 min | 55 °C for 45 s | |
Extension | 72 °C for 80 s | 72 °C for 1 min | |
End of cycle | |||
 Final extension | 72 °C for 10 min | 72 °C for 5 min | |
 Amplicon size |  ~ 1500 bp |  ~ 600 bp | |