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Title: RAPID, SPECIFIC AND QUANTITATIVE ASSAYS FOR THE DETECTION OF THE ENDOPHYTIC BACTERIUM METHYLOBACTERIUM MESOPHILICUM IN PLANTS

Author
item LACAVA, P.T. - U. SAO PAULO
item LI, WENBIN - U. FLORIDA
item Hartung, John

Submitted to: Journal of Microbial Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/19/2005
Publication Date: 11/2/2005
Citation: Lacava, P., Li, W., Hartung, J.S. 2005. Rapid, specific and quantitative assays for the detection of the endophytic bacterium methylobacterium mesophilicum in plants. Journal of Microbial Methods. 65:535-541.

Interpretive Summary: Xylella fastidiosa is a pathogenic bacterium that causes citrus variegated chlorosis (CVC) disease in South and Central America. The pathogen colonizes internal plant tissues, and previous work with CVC disease has considered Xylella fastidiosa alone. However, recent studies have shown that other bacteria, notably Methylobacterium mesophilicum, can colonize the same internal sweet orange plant tissues, without causing disease symptoms. Until now, there have not been adequate methods available to study any possible dynamic interactions between the pathogen, X. fastidiosa, and Methylobacterium mesophilicum, because these bacteria are difficult to isolate and very slow growing in culture. We have previously developed specific, rapid and quantitative assay methods for X. fastidiosa. We now report the development of similar specific, rapid and quantitative assays for Methylobacterium mesophilicum. Our results show that both Xylella (the pathogen) and Methylobacterium (a non pathogen) can both be detected and quantified in a matter of hours. Our preliminary results also show that when M. mesophilicum is present in plants at the same time as the pathogen, Xylella, the population levels of X. fastidiosa are lower. Our methods will be useful to scientists interested in the interactions of Xylella fastidiosa and other bacteria in diseased plants.

Technical Abstract: Xylella fastidiosa is a xylem-limited bacterium that causes citrus variegated chlorosis disease in sweet orange. There is evidence that Xylella fastidiosa interacts with endophytic bacteria also resident in the xylem of sweet orange, and that these interactions, particularly with Methylobacterium mesophilicum may affect disease progress. However, efficient methods for detection and enumeration of these bacteria in planta were lacking. We have previously developed standard and quantitative PCR-based assays specific for X. fastidiosa using the LightCycler® system, and now report the development of both standard and quantitative PCR assays for Methylobacterium mesophilicum. The assays are specific for Methylobacterium mesophilicum and do not amplify DNA from other species of Methylobacterium or other bacteria commonly associated with citrus or plant tissue. Other bacteria tested included Curtobacterium flaccumfaciens, Pantoea agglomerans, Enterobacter cloacae, Bacillus sp., Xylella fastidiosa, Xanthomonas axonopodis pv. citri, and Candidatus Liberibacter asiaticus. We have demonstrated that with these methods we can quantitatively monitor the colonization of xylem by M. mesophilicum during the course of disease development in plants artificially inoculated with both bacteria.