Xanthomonads on Tomato and Pepper
Sample preparation
Symptomatic plant tissue should be cut into fine strips using sterile technique, placed onto the
glass slide in the kit and observed for bacterial streaming. This may then be used directly for
PCR. If little streaming is seen, place tissue strips in a sterile epi-tube with sterile water and
allowed to sit at room temp for 30 minutes. Streak sample suspension onto Nutrient Agar and
grow 24-48 hrs. Use 1 mL sterile nanopure water to wash over the cells on the plate. Collect
with pipette and place into a sterile eppitube. Use a 1/10 dilution of cells for the PCR.
Rxn Master Mix (25 µL tube)
Sterile Nanopure water
2X Brilliant SYBR Green QPCR Master Mix
SmartCycler Additive Reagent (SAR)
Primer 1…RST 65
Primer 2…RST 69
4.0 µL
12.5 µL
2.5 µL
2.5 µL
2.5 µL
Sample
1 µL of either ooze from diseased tissue or cell suspension harvested from petri dishes (or
controls as noted below).
Primers
Prepare to a concentration of 1.5 µM (1.5 pmol/µL)
RST 65 5’-GTC GTC GTT ACG GCA AGG TGG TCG -3’
RST 69 5’-TCG CCC AGC GTC ATC AGG CCA TC -3’
Controls
Negative control:
Positive control #1:
Positive control #2:
1 µL sterile Nanopure water
1 µL cell suspension of one of the known xanthomonad isolates (75-3,
XV-56, 91-118, or GA-2)
1 µL cell suspension one of the known xanthomonad isolates plus 1 µL
of test sample. If results show evidence of inhibition, retest sample
extract at 10-fold dilution.
Cycling protocol
Name of protocol: Xanthomonas
1. 95C
2. 40 cycles
600 sec
95C
63
74
30 sec
30
45
optics off
off
on
95C
63
74
30
30
300
optics off
off
on
3. 1 cycle
4. Melt Curve
Start 60C
end 95
Deg f 0.2
Duration of protocol: 1 hr 28 min.
Results expected
Product of 420 bp long with a Tm of 88.0-91.0. (Tm values for GA-2 are approximately 1°C
higher than the other positive controls. Template-free controls in this assay have a Tm of 76.379.6. This protocol has been developed using whole-cell suspensions of each of the four groups
of xanthomonads from tomato and pepper (as defined in Jones et al. 2004):
Group A (isolate 75-3): Xanthomonas euvesicatoria
Group B (isolate XV-56): Xanthomonas vesicatoria
Group C (isolate 91-118): Xanthomonas perforans
Group D (isolate GA-2): Xanthomonas gardneri
The RST 65/69 primers have been shown by Cuppels et al (2006) to give positive reactions to a
number of pathogenic Xanthomonas species and pathovars from other hosts, indicating less than
complete specificity for bacterial spot-producing strains. However, they reacted negatively to
non-pathogenic epiphytic Xanthomonads isolated from tomato and pepper (Cuppels et al., 2004;
Obradovic et al., 2004). Obradovic et al (2004) found that a low percentage of pathogenic
isolates did not react positively to the RST 65/69 primer pair.
Using pure cultures, no non-specific products are produced. Detection of groups B and D is less
sensitive than groups A and C. The melt curve for isolate XV-56 and GA-2 is slightly
asymmetrical.
Groups can be distinguished by digestion of PCR product with HaeIII, Taq1, and Cfo1 restriction
enzymes; see Obradovic et al, 2004.
Notes
The RST 65/69 primers amplify a conserved portion of a hrp gene cluster.
Brilliant SyBr Green Master Mix cannot be re-cycled after an initial PCR cycling run; probably
due to limited enzyme stability at high temps. New reactions need to be set up for further testing.
References
Cuppels, D. A., Louws, F. J., and Ainsworth, T. 2006. Development and evaluation of PCR-based
diagnostic assays for the bacterial speck and bacterial spot pathogens of tomato. Plant Dis.
90:451-458.
Obradovic, A., Mavridis, A., Rudolph, K., Janse, J. D., Arsenijevic, M., Jones, J. B., Minsavage,
G. V., and Wang, J.-F. 2004. Characterization and PCR-based typing of Xanthomonas
campestris pv. vesicatoria from pepper and tomatoes in Serbia. Eur. J. Plant Pathol. 110:285-292.
(presents primer sequences and typical results)
Jones, J. B., Lacy, G. H., Bouzar, H., Stall, R. E., and Schaad, N. W. 2004. Reclassification
of the xanthomonads associated with bacterial spot disease of tomato and pepper. Syst. Appl.
Microbiol. 27:755-762.
Jones, J. B., R. E. Stall, and H. Bouzar. 1998. Diversity among xanthomonads pathogenic on
pepper and tomato. Annu. Rev. Phytopathol. 1998. 36:41–58
Leite, et al. 1994. Detection and identification of phytopathogenic Xanthomonas strains by
amplification of DNA sequences… Appl. Envir. Microbiol. 60:1068-77.
Document Control
2/13/2016
Suggested RFLP Enzyme recipes for Xanthomonads
amplicons @~ 0.4 µg/rxn
Amplicon clean-up using Qiagen PCR Purification Kit (cat. 28004):
Use elution volumes of 15µl of a 100µl PCR rxn product.
HaeIII (NEB) 20µl rxn, NEB Buffer 2
10x NEB Buffer 2
Amplicon, cleaned
Sterile Nanopure water
HaeIII enzyme, 5U
2.0µl
3.0µl
14.5µl
0.5µl
Run @ 37ºC; 1 hr
Taqα1 (NEB) 20µl rxn, NEB Buffer 3
10x NEB Buffer 3
Amplicon, cleaned
BSA, 100x
Sterile Nanopure water
Taqα1 enzyme, 5U
2.0µl
3.0µl
0.1µl
14.65µl
0.25µl
Cfo1 (Promega) 20µl rxn, Promega Buffer B
10x Promega Buffer B
Amplicon, cleaned
BSA (Promega, 10µg/µl)
Sterile Nanopure water
Cfo1 enzyme, 5U
Run @ 65ºC; 1 hr
Run @ 37ºC; 1 hr
2.0µl
3.0µl
1.0µl
13.5µl
0.5µl
Load 10 µl of a reaction/lane onto a 3% NuSieve agarose gel with 0.5% TBE buffer.