Assay for growth of Pseudomonas syringae pv. tomato in tomato leaves
Developed by Pete E. Pascuzzi, Jeffrey C. Anderson, and Gregory B. Martin
Boyce Thompson Institute for Plant Research
12 November 2004
Bacterial strains are streaked from glycerol stocks onto King's B (KB) medium plates containing
rifampicin (100 mg/L) and kanamycin (25 mg/L) and incubated at 30C for 3 days. Bacteria are
then resuspend in liquid King's B (KB) medium to an OD600 of 1.0, and 0.1 ml of this suspension
is spotted onto a fresh KB plate as above and incubated overnight at 30C. These cells are
resuspend in 0.1 M sucrose containing 10 mM MgCl2 to an OD600 of 0.1. This is used to
inoculate 4 L of 10 mM MgCl2 containing 0.002% Silwet to a final OD600 of 5 x 10-5. This
produces an inoculum of 2 x 104 cfu/ml for Pst T1 strains as confirmed by plating serial dilutions
and counting the resulting colonies.
Tomato plants used for the assays are transplanted to 4" pots one week before infiltration.
Two days before infiltration, plants are moved to a climate-controlled greenhouse that is
maintained at an average temperature of 23C with a 16 hour day length. Plants are infiltrated 4
weeks after seed sowing by totally immersing the plants in inoculum and subjecting them to a 2
minute vacuum with a pump rated at 10 torr and 29.3 L/min. After air-drying, plants are returned
to the greenhouse.
Bacterial growth is monitored over several days with samples typically taken on days 0, 2,
3, and 4. For Pst T1, bacterial growth usually reaches its maximum on day 3 or 4, depending on
the virulence of the strain and greenhouse conditions. Each sample consists of 3 1-cm diameter
discs taken from one leaflet with two leaves sampled per plant. Discs are floated abaxial side
down on 1 ml of 0.1 M sucrose containing 10 mM MgCl2 and incubated at 12C and 125 rpm for
4 hrs. 10-fold serial dilutions in the same buffer are made, and 20 l of these dilutions are
spotted onto KB plates with antibiotics as above. Colonies were counted 48 to 60 hours after
plating with the aid of a dissecting microscope.
We have found that consistent handling of bacterial strains and plants, as well as the use
of a climate controlled greenhouse, to be critical factors in producing consistent and reproducible
results.