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Copper tolerance in Pseudomonas
syringae pv. tomato from tomatoes in
Queensland, Australia
Karina Griffin, Cherie Gambley & Philip Brown
These slides will be made available on the APPS website
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Study Overview
Pseudomonas syringae pv. tomato = Bacterial Speck Disease
Control reliant on Copper based products
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Study Overview
Intensive copper use in crops
Risk of copper tolerance
development
Environmental impacts
Copper tolerance in P. syringae pv.
tomato reported globally, 2
Australian studies
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Study Overview
Sampled commercial
tomato crops 2015-2017
101 plant samples
31 P. syringae pv. tomato
isolates
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Shameless self promotion…
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in vitro copper tolerance screening
Methodology testing: Effect of media type and pH on
in vitro results (Paper)
All isolates copper tolerant – MIC 0.8-1.5 mM on CYEG
(Casitone yeast extract glycerol agar)
Confirmed in vivo
Herbarium isolates from 1970-80, also
copper tolerant
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PCR to identify cop genes
Plasmid encoded cop
genes linked to copper
tolerance
Normal metabolism requires
low levels of copper ions –
chromosomally encoded
Initial focus on copAFigure extracted from Cooksey 1994
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Isolate ID/sP. syringae
pv. Source
MIC (mM)
Copper status
Primers
producing a
fragment
No. Cu binding
motifs (Cha & Cooksey 1991)
*M19930
*JQ418536tomato USA N/A Tolerant N/A 4
*KY362372
*CP000075syringae
Spain 2016
USA 2005N/A Tolerant N/A 4
*AE016853
tomato
strain
DC3000
UK
19600.5 Sensitive None 3
63.1 tomato This Study 1.5 TolerantPCCop,
KGCop1&24
16 study
isolatestomato This Study 0.8-1.0 Tolerant KGCop1 3
BRIP38744BRIP38746
tomatoAust.
1970-801.5 Tolerant PCCop ?
Primer details
PCCop = Almost entire copA KGCop2 = 1st half of copA KBCop1 = 2nd half of copA
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DC3000 Cu Sensitive
M19930 Cu Tolerant (USA ref)
1.1 Cu Tolerant (This Study)
52.2 Cu Tolerant (This Study)
94.1 Cu Tolerant (This Study)
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63.1 100% identity* to M19930 reference (nt and aa)
Rest of study isolates ~65% nt identity* & ~70% aa identity*
*273 aa & 842 nt region
M19930 Cu Tolerant (USA ref) – 4 motifs
63.1 Cu Tolerant (This Study) – 4 motifs
1.1 Cu Tolerant (This Study) – 3 motifs
94.1 Cu Tolerant (This Study) – 3 motifs
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Conclusions
P. syringae pv. tomato from QLD, NSW & VIC are tolerant
to copper
Study isolates carry the copA gene
Sequence differences in the copper binding region
Genome sequences
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Acknowledgments
Supervisors Cherie (QLDDAF), Phil & Jady (CQU)
Paul Campbell (QLDDAF)
QLDDAF Research staff at the Ecosciences Precinct Brisbane
& Applethorpe research station
Industry co-operators for field sampling locations
Scholarship & Research Funding: Australian Government
Research Training Program (RTP) Scholarship & CQU
Various reference cultures:
Karren Gowan: Agricultural Scientific Collections Trust, New
South Wales Australia (ASCT)
Roger Shivas and Yu Pei Tan: The Plant Pathology Herbarium of
Biosecurity Queensland Department of Agriculture and Fisheries
Eric Newberry & Matthews Paret: University of Florida USA
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References
Cha, J.-S., & Cooksey, D. A. (1991). Copper resistance in Pseudomonas syringae
mediated by periplasmic and outer membrane proteins. Proceedings of the
National Academy of Sciences, 88(20), 8915-8919.
Cooksey, D. A. (1994). Molecular mechanisms of copper resistance and
accumulation in bacteria. FEMS microbiology reviews, 14(4), 381-386.
Griffin, K., Gambley, C., Brown, P., & Li, Y. (2017). Copper-tolerance in
Pseudomonas syringae pv. tomato and Xanthomonas spp. and the control of
diseases associated with these pathogens in tomato and pepper. A systematic
literature review. Crop Protection, 96, 144-150.
doi:http://dx.doi.org/10.1016/j.cropro.2017.02.008
Roberts, S. A., Weichsel, A., Grass, G., Thakali, K., Hazzard, J. T., Tollin, G.,
Montfort, W. R. (2002). Crystal structure and electron transfer kinetics of CueO, a
multicopper oxidase required for copper homeostasis in Escherichia coli.
Proceedings of the National Academy of Sciences, 99(5), 2766-2771.