Colonization of Tomato Stem

Written/Edited by: Tiffany Lowe-Power, Madeline Hayes, Mandel lab

Experimental Design:

Note: This is the protocol where 1 strain is inoculated per plant. Sometimes we want to do competitive inoculations where 2 strains are co-inoculated. For those, see the competition protocol.

• Timing:
  • Colonization assays (e.g. comparing two strains) should be sampled at specific time points (will show delay(s) in colonization if time points are before population levels saturate) and not at specific symptom level (even if a strain takes longer to initiate symptoms, at symptom onset, plants will have ~5x10⁸-5x10⁹ CFU/g)
  • Growth curve after tomato petiole inoculation: 0, 1, 2, 3 dpi
    • Sample size, for time 0, N=3 per condition (plant genotype/strain/etc) is sufficient because there shouldn’t be much variation here. For other time points, larger N is better. Aim for at least N = 8 for other time points.
    • Note, it’s recommended that you transplant extra plants in case you mess up your inoculation.
  • Growth curve (stem) after soil drench inoculation: 4, 6, 8 dpi
    • Time 0 is not informative because bacteria are sampled into the soil and sampling is in the stem.
    • Infection via soil-drench is stochastic. 4 dpi is probably the earliest you want to do, but even then, many plants will be uncolonized.
    • Even though you will probably plan days 1-3 over a weekend, it is crucial to keep soil moisture level high during this window.
• Sampling site:
  • Cut-Petiole: Sample at the site of inoculation '+0'and a distal site (e.g. 2 cm below the site of inoculation '-2').
    • After cut-petiole, the bacteria will first establish at the site of inoculation, then below the site of inoculation before establishing above the site of inoculation. This probably reflects connectivity of vascular architecture
  • Soil-Soak: Sample at the cotyledon junction

Plant Inoculation

Follow the petiole or soil soak inoculation protocols

Destructive Sampling of Stem

Preparation

1. CPG+TZC(+antibiotic) plates: 1 per sampling site; labeled for 10⁰ to 10^-7 dilutions.
2. Bead tubes: 1 per sampling site; filled with 4x 2.22 mm metal beads & 900 ul dI H2O;
   • labeled on side–powerlyzer rubs labels off top
   • Can prepare up to 1 week in advance
   • Major tip: Use a pipette tip to quickly load beads into tubes: https://www.reddit.com/r/labrats/comments/59ojh8/glass_bead_dispenser/d9an6ge/
3. Check consumable levels in advance:
   • 200 ul tips (1 box per 12 samples) - autoclaved
   • 96-well round-bottom microplate (1 per 12 samples) - sterilized in 10% bleach 30 min, 4x thorough rinses with diH2O, dried in 50C oven… or new microplate.
4. Load microplate with di H2O to dilution plate samples
   • Pour di H2O into a sterile reservoir (autoclaved glass petri dish or plastic petri dish).
   • Using multichannel P200, add 180 ul to 7 rows well of microplate (A-G).

Sampling

1. Use a razor blade to sample 100 mg +/- 10 mg stem. Record mass for normalization.
   • Sharps safety: Discard dulled blades in sharps trash. When cutting stem, especially of older lignified plants, be careful of hand placement with respect to cutting angle.
2. Place plant stem in bead tube. Homogenization will be inefficient for biomass that does not move readily; cut large samples before homogenization. Repeat for 12-24 samples.
3. Homogenize samples in batches of 12-24. Verify lid is screwed on each tube as you load it
   • Powerlyzer program: 2 rounds of 2200 rpm for 1.5 min each with 4 min rest (to minimize sample heating).
   • If you have multiple treatments (e.g inoculating strains) and will process in multiple batches, evenly spread out treatments between batches.
   • If you need to pause, it’s best to pause before homogenization. Homogenization will release nutrients that can promote bacterial growth before dilution plated.
4. After homogenization, inspect samples to verify complete homogenization. Protip: incompletely homogenized samples usually have less chlorophyll in suspension.
5. Dilute samples according to dilution plating protocol. Finish homogenizing samples in batches of 12 or 24.

Data Analysis:

• Copy calculations from bacterial density workbook into a digital lab notebook entry and calculate CFU / g stem.
• In GraphPad Prism, plot individual data points and medians on a logarithmic scale.
• The data are often not normally distributed, with different variances, and the outliers may contain biologically meaningfully information. Therefore, non-parametric tests provide a useful method to determine whether the treatments differ significantly.
• Use GraphPad Prism software or similar for statistical analysis. For two treatments, use the Wilcoxon Rank Sum test. For comparisons among greater than two treatments, use the Kruskal-Wallis test with appropriate post-tests for multiple comparisons.
• Recommendation: Keep track of experiment throughout digital files with a timestamp on the inoculation day. e.g 20191231 (year, month, day is sequential in computer and compatible for US vs. International dating norms)

Advice

(1) After running the powerlyzer, visually inspect each tube to ensure that the tissue was homogenized. Especially when stems are rigid (i.e. older tomatoes), they become more difficult to grind. I inspect as I move tubes from the powerlyzer to my tube-rack. As I do this, I tilt the tube so that the liquid goes from top to bottom, and I watch for plant chunkies. If there are large chunks, then I run the sample for a second homogenization. Do be careful to not over-heat samples with repeated homogenization cycles. The moving beads = kinetic energy = heat generation. It might kill Ralstonia if it rises too high (i.e. probably above 50C if I recall…)