Rapid, specific and quantitative assays for the detection of the endophytic bacterium Methylobacterium mesophilicum in plants

• Published in: Journal of microbiological methods Vol. 65; no. 3; pp. 535 - 541
• Main Authors: Lacava, P.T., Li, W.B., Araújo, W.L., Azevedo, J.L., Hartung, J.S.
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier B.V 01-06-2006; Elsevier Science
• Subjects: Bacillus; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Citrus; Citrus sinensis; Citrus sinensis - microbiology; Curtobacterium flaccumfaciens; Diverse techniques; DNA Primers; DNA, Bacterial - analysis; DNA, Intergenic - analysis; Endophyte; Enterobacter cloacae; Fundamental and applied biological sciences. Psychology; LightCycler; Methylobacterium; Methylobacterium - classification; Methylobacterium - genetics; Methylobacterium - isolation & purification; Methylobacterium mesophilicum; Microbiology; Molecular and cellular biology; Pantoea agglomerans; Plant Diseases - microbiology; Polymerase Chain Reaction - methods; Real-time quantitative PCR; RNA, Ribosomal, 16S - genetics; Sensitivity and Specificity; Systematics; Time Factors; Xanthomonas axonopodis; Xylella - genetics; Xylella - isolation & purification; Xylella fastidiosa; Citrus; Real-time quantitative PCR; Endophyte; LightCycler; Xylella fastidiosa; Plant pathogen; Citrus fruit; Rutaceae; Real time; Polymerase chain reaction; Dicotyledones; Angiospermae; Spermatophyta; Detection; Quantitative analysis
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2005.09.015

Xylella fastidiosa is a xylem-limited bacterium that causes citrus variegated chlorosis disease in sweet orange. There is evidence that
X. fastidiosa interacts with endophytic bacteria present in the xylem of sweet orange, and that these interactions, particularly with
Methylobacterium mesophilicum, may affect disease progress. However, these interactions cannot be evaluated in detail until efficient methods for detection and enumeration of these bacteria in planta are developed. We have previously developed standard and quantitative PCR-based assays specific for
X. fastidiosa using the LightCycler® system [Li, W.B., Pria Jr., L.P.M.W.D., X. Qin, and J.S. Hartung, 2003.Presence of
Xylella fastidiosa in sweet orange fruit and seeds and its transmission to seedlings. Phytopathology 93:953-958.], and now report the development of both standard and quantitative PCR assays for
M. mesophilicum. The assays are specific for
M. 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.,
X. 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.