Procedures for Bacterial Spot Inoculation

Reagents

Yeast Dextrose Calcium Carbonate Agar (YDC)

1. Combine reagents and autoclave (121 °C, 1 Kg/cm²)
2. Separately autoclave 30% glucose (20.1 g glucose in 67 ml ddH₂0)
3. Combine flasks after autoclaving, cool, then pour in plates

Potassium Phosphate Buffer (KPB) pH 7.4, 10 mM

1. Combine reagents and autoclave
2. Autoclave double distilled (dd) H₂O

Methods

Prepare inoculum by growing bacteria on YDC agar media at 28 °C for 48–72 h (Fig. 1). Wash bacterial cells from agar plates and suspend in sterile 10 mM Potasium Phosphate Buffer (KPB, pH 7.4) . Standardize suspensions to A600 = 0.15 (coresponds to a concentration of approximately 3 X 10⁸ CFU/ml) (Fig. 1).

Figure 1. Petri dish with Xanthomonas (left); suspension of bacteria and loading syringe (center); inoculation (right). Photo credits: Hui Wang, The Ohio State University.

For field trials, plants are inoculated as four-week-old seedlings in the greenhouse one week prior to transplanting into the field. Plants are sprayed to run-off with a fine mist using a suspension of A600 = 0.15 bacteria using a Preval Spray Gun (Preval Sprayer Division, Yonkers, NY).

Additional Resources

• Chakravarthy, S., A. C. Velásquez, and G. B. Martin. 2009. Assay for pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) in plants. [Online video]. Journal of Visualized Experiments 31. Available at: http://dx.doi.org/10.3791/1442 (verified 01 Mar 2012).
• Schaad, N. W., J. B. Jones, and W. Chun. 2001. Laboratory guide for identification of plant pathogenic bacteria. Third Edition. APS Press, The American Pytopathological Society, St. Paul, MN.

Funding Statement

Development of this lesson was supported in part by the National Institute of Food and Agriculture (NIFA) Solanaceae Coordinated Agricultural Project, agreement 2009-85606-05673, administered by Michigan State University. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the United States Department of Agriculture.

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