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A Technique for Precise Inoculation of the Internal Phyllosphere of Cotton with Xanthomonas campestris pv. malvacearum

October 2003 , Volume 93 , Number  10
Pages  1,204 - 1,208

N. Kangatharalingam , Margaret L. Pierce , and Margaret Essenberg

Department of Biochemistry & Molecular Biology, Oklahoma State University, Stillwater 74078

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Accepted for publication 9 April 2003.

A technique was developed to inoculate uniformly and gently the internal phyllosphere from the upper surface of cotton leaves with the phytopathogenic bacterium Xanthomonas campestris pv. malvacearum. The inoculum consisted of 2 to 3 × 107 CFU/ml in CaCO3-saturated sterile distilled water containing 0.02%, vol/vol, of the wetting agent Silwet L-77. A custom-made inoculation apparatus was employed to immerse a circular area of the adaxial surface of a leaf in inoculum for 90 s. This resulted in uniform, passive entry of bacteria into the substomatal chambers, producing an endophytic bacterial population of 2 × 104 CFU/cm2. Microscopic signs of infection were visible 48 to 72 h after inoculation. In susceptible leaves, uniformly distributed water-soaked spots were observed 7 to 8 days after inoculation. When the technique was used on resistant leaves, the autofluorescence that is characteristic of hypersensitively necrotic cells developed in the guard cells and palisade cells lining substomatal chambers, but not in the underlying spongy mesophyll.

Additional keyword: adaxial epidermis.

© 2003 The American Phytopathological Society