OSU Logo

JOHNSON LAB CREATES FAST MOLECULAR DETECTION TOOL FOR BACTERIAL PATHOGEN IN CARROT

Temple, T. N., du Toit, L. J., Derie, M. L., and Johnson, K. B. 2013. Quantitative molecular detection of Xanthomonas hortorum pv. carotae in
carrot seed before and after hot-water treatment. Plant Dis. 97:1585-1592.

Molecular assays to detect and quantify DNA from viable cells of the
seedborne pathogen Xanthomonas hortorum pv. carotae in carrot seed
were developed and evaluated for use on nontreated and hot-watertreated
seed lots. Both a TaqMan real-time polymerase chain reaction
(PCR) assay and a loop-mediated isothermal amplification (LAMP)
dilution endpoint assay detected and quantified DNA from viable
pathogen cells after treatment of carrot seed washes with the live-dead
discriminating dye propidium monoazide (PMA). The detection limits
of the assays were approximately 101 CFU for pure cultures of X. hortorum
pv. carotae, and 102 to 103 CFU/g seed from naturally infested
carrot seed lots. X. hortorum pv. carotae in and on carrot seed was
killed by soaking the seed in hot water (52°C for 25 min), and a subsequent
PMA treatment of these hot-water-treated seed washes suppressed
detection of the pathogen with both the real-time PCR and
LAMP assays. For 36 commercial seed lots treated with PMA but not
hot water, regression of colony counts of X. hortorum pv. carotae
measured by dilution plating on a semiselective agar medium versus
estimates of pathogen CFU determined by the molecular assays resulted
in significant (P ≤ 0.05) linear relationships (R2 = 0.68 for the
real-time PCR assay and 0.79 for the LAMP assay). The molecular
assays provided quantitative estimates of X. hortorum pv. carotae
infestations in carrot seed lots in <24 h, which is a significant improvement
over the 7 to 14 days required to obtain results from the traditional
dilution-plating assay.

LAMP

For each picture above (and on the front page) there is a positive LAMP reaction in the amplification tube on the left and a negative LAMP reaction on the right.  On the left a positive reaction is a yellow color and on the right a fluorescent green color (addition of Pico green to the reaction tube). Negative reactions are shown as pink color or non-fluorescent.

full article