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ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Subtropical Plant Pathology Research » Research » Publications at this Location » Publication #187230
Title: USE OF THE GREEN FLUORESCENT PROTEIN (GFP) TO STUDY SURVIVAL OF XANTHOMONAS AXONOPODIS ON CITRUS PLANT SURFACES

Author
item CUBERO, J. - INIA, MADRID, SPAIN
item GRAHAM, J. - UNIV. OF FLORIDA
item REDONDO, A. - UNIV. OF FLORIDA
item DEKKERS, M. - UNIV. OF FLORIDA
item ZHANG, Y. - UNIV. OF FLORIDA
item JONES, J. B. - UNIV. OF FLORIDA
item WILSON, M. - COLORADO COLLEGE

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 9/30/2005
Publication Date: 11/30/2005
Citation: Cubero, J., Graham, J., Redondo, A., Dekkers, M., Zhang, Y., Jones, J., Wilson, M. 2005. Use of the green fluorescent protein (gfp) to study survival of xanthomonas axonopodis on citrus plant surfaces. Abstract. International Citrus Canker and Huanglongbing Workshop, Orlando FL, November 7-11, 2005.

Interpretive Summary:

Technical Abstract: Although much research has been conducted on survival of Xanthomonas axonopodis pv. citri (Xac) as an epiphyte some important issues remain unresolved. Xac can survive on non-symptomatic leaves for short periods (hours) as an epiphyte depending on the relative humidity conditions the leaves are subjected to under drying conditions (Graham et al., 2004). This capability for short-term survival is not considered a serious risk for Xac to initiate new infections. However, previous studies of population dynamics on symptomatic or non-symptomatic plants were based on bacterial isolation on culture media, and therefore only viable and culturable cells were detected. Viable but non-culturable (VBNC) is a physiological condition described for many bacterial species and for some plant pathogenic bacteria including Xanthomonas. The epidemiological significance of VBNC is unknown because accurate identification of viable bacteria in planta is problematic. Transformation of the bacterium with the green fluorescent protein gene (gfp) gene can be used as the reporter system to locate the bacterium in or on the plant surface in situ by fluorescence and confocal microscopy. The plasmid transformants of X. axonopodis pv. citrumelo with gfp maintain virulence as compared with wild type strain. However, the GFP marker is too stable to monitor bacterial survival because dead bacterial cells still fluoresce. Instead an unstable gfp tranformant of X. axonopodis pv. citrumelo has been constructed to demonstrate the survival of this bacterium. The unstable Gfp variant is produced by the addition of an amino acid tail to the 3’ terminal of the gfp sequence to render the protein susceptible to proteases within the bacterium (Andersen et al., 1998). Hence, the living bacterium synthesizes and degrades the Gfp protein simultaneously and only cells that actively regenerate the gene product exhibit fluorescence. X. axonopodis pv. citrumelo isolates transformed with native and labile Gfp were sprayed on or infiltrated into Swingle citrumelo leaves. Bacteria survive on leaf surface when applied with a phosphate buffer osmoregulant, but die faster under low humidity conditions when applied to the leaf surface with rainwater only. Bacterial aggregation is greater in the presence of the phosphate buffer. The osmoticum appears to be responsible for the maintenance of humidity around the aggregated colonies on the leaf surface. In planta, bacteria remain viable when located in the intercellular space proximal to the stomatal chamber. The gfp system developed with X. axonopodis pv. citrumelo serves as a model for further studies of X. axonopodis pv. citri survival in planta and on the surfaces of citrus fruits under environmental conditions relevant to field and post harvest conditions, respectively.