Pseudomonas syringae pv. tomato
Sample preparation
This protocol has not been tested with plant material. Proposed plant sampling should utilize the
Sampling Kit for Bacterial Vascular Wilts listed in the front of this notebook. The plant tissue
should be cut into fine strips using sterile techniques, placed onto the glass slide in the kit and
observed for bacterial streaming. This may then be used directly for PCR. If little streaming is
seen, place tissue strips in a sterile eppitube with sterile water and allowed to sit at room temp for
30 minutes. Streak sample suspension onto Nutrient Agar and grow 24-48 hrs. Use 1 mL sterile
nanopure water to wash over the cells on the plate. Collect with pipette and place into a sterile
eppitube. Use a 1/10 dilution of cells for the PCR.
Rxn Master Mix (25 µL tube)
Sterile Nanopure water
SmartCycler Additive Reagent (SAR)
Primer 1…Pst 1
Primer 2…Pst 2
2X Power SyBr Green Master Mix
4.0 µL
2.5 µL
2.5 µL
2.5 µL
12.5 µL
Primers
Prepare to a concentration of 1.5 µM (1.5 pmol/µL)
Pst 1 5’-GGC GCT CCC TCG CAC TT -3’
Pst 2 5’-GGT ATT GGC GGG GGT GC -3’
Controls
Negative control:
1 µL sterile Nanopure water
Positive control #1:
1 µL Pseudomonas syringae pv tomato cell suspension
Positive control #2:
1 µL Pseudomonas syringae pv tomato cell suspension Plus 1 µL of test
sample. If results show evidence of inhibition, retest sample extract at 10-fold dilution.
Cycling protocol
Name of protocol: Pseudo syrin pv tom
1. 95C
2. 40 cycles
600 sec
95C
55
74
30 sec
60
120
74
120
optics off
off
on
3. Hold
4. Melt Curve
Start 60C
end 95
Duration of protocol: 2 hr 46 min.
off
Deg f 0.2
Results expected
Product of 650 bp long with a Tm of 88.0-89.0. Template-free controls in this assay have a Tm of
76.3-79.6. This protocol has been developed using seven whole-cell suspension isolates of
Pseudomonas syringae pv tomato obtained from OSU. Using pure cultures, no non-specific
products are produced. This protocol has yet to be tested against suspect plant tissue (may 27,
2004).
Notes
Control cell isolates Pto 36 and Pto 177 consistently do not amplify or are inconsistent. These are
probably coronatine non-producers.
Control isolate DC3000 or isolate 23.20 are the BEST to use as a positive control.
(November 5, 2009)