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A Simple Method to Monitor Growth of Bacterial Populations in Leaf Tissue. Greg Bertoni, Department of Botany and Plant Pathology, Oregon State University, Corvallis 97331; Dallice Mills, Department of Botany and Plant Pathology, Oregon State University, Corvallis 97331. Phytopathology 77:832-835. Accepted for publication 4 December 1986. Copyright 1987 The American Phytopathological Society. DOI: 10.1094/Phyto-77-832.

This paper describes a simple, reproducible, and inexpensive technique to accurately monitor the growth rates of phytopathogenic bacteria in plant leaves. After growing bacterial cultures to 108 colony-forming units (cfu) per milliliter and diluting 100-fold in phosphate buffer, a disposable syringe was used to consistently introduce approximately 103 cfu into discrete sites on the leaf blade for subsequent random sampling on days 0, 1, 3, and 6 postinoculation. Leaf disk samples containing the inoculated area were then taken with a 6-mm cork borer, macerated in a unique tissue grinding apparatus devised for rapid and efficient isolation of bacteria from these samples and bacterial populations per sample determined using a microplating method that required a minimum of time and materials. This technique was used successfully in bean leaves to compare the in planta growth dynamics of two wild-type strains of Pseudomonas syringae pv. syringae, the causative agent of brown spot disease of Phaseolus vulgaris, and two mutant strains of lessened virulence derived after Tn5 mutagenesis of one of the wild-type strains. A clear difference in growth rates and plateau populations, correlating with symptom severity, was observed between the two wild-type strains, and altered growth patterns compared with the parental were demonstrated for the two mutant strains.