Session 2: Liberibacter solanacearum comparative genomics and diagnostics
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Liberibacter solanacearum comparative genomics and diagnostics
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Session 2: Liberibacter solanacearum comparative genomics and diagnostics
- 1. biosecurity built on science Liberibacter solanacearum comparative genomics and diagnostics Creswick, Science Exchange, 26-28th Oct 2016 Sarah Thompson, Chris Johnson, Rebekah Frampton, Kerry Sullivan, Charles David, Falk Kalamorz, Neil Gudmestad, Grant Smith.
- 2. biosecurity built on science Introduction to Liberibacter solanacearum • Alpha-proteobacteria discovered in 2008 • Phloem-limited in plants • Vectored by psyllids • 5 haplotypes described • A – USA, NZ • B – USA • C – Scandanavia • D – Europe • E - Europe
- 3. biosecurity built on science Liberibacter solanacearum haplotypes A and B C D and E
- 4. biosecurity built on science Liberibacter solanacearum haplotypes A A and B C D and E
- 5. biosecurity built on science Implications for Biosecurity Name-based biosecurity Increased importance of defining names PB-CRC2002/PB-CRC2156 Genome-informed diagnostics • Gain a better understanding of Lso taxonomy • Improve the current diagnostic for Lso by including a non rRNA target • Develop assays to differentiate the solanaceous-infecting haplotypes from the rest. Increased importance of sub-species differentiation e.g. Virulence (Psa) Hosts (Liberibacter)
- 6. biosecurity built on science ANI comparison of the draft genomes Genus Species Subspecies ANI 95% CLsoA_NZ1 CLsoB_ZC1 CLsoD_Is CLsoE_CI2 CLsoA_NZ1 100 CLsoB_ZC1 97.69 100 CLsoD_Is 98.29 97.71 100 CLsoE_CI2 97.77 97.38 98.68 100
- 7. biosecurity built on science ANI comparison of the draft genomes Genus Species Subspecies ANI 95% CLsoA_NZ1 CLsoB_ZC1 CLsoD_Is CLsoE_CI2 CLsoA_NZ1 100 CLsoB_ZC1 97.69 100 CLsoD_Is 98.29 97.71 100 CLsoE_CI2 97.77 97.38 98.68 100 Benefit to biosecurity workers: Moving towards identifying at-risk sectors or regions for emerging diseases
- 8. biosecurity built on science Table 3. Cycle threshold values for screen of 15 qPCR assays against 43 samples Thompson, S. M., Johnson, C. P., Lu, A. Y., Frampton, R. A., Sullivan, K. L., Fiers, M.W. E. J., Crowhurst, R. N., Pitman, A. R., Scott, I. A.W., Wen, A., Gudmestad, N. C., and Smith, G. R. 2015. Genomes of ‘Candidatus Liberibacter solanacearum’ haplotype A from New Zealand and the United States suggest significant genome plasticity in the species. Phytopathology 105:863-871. Sample Clade Host Collected Year LsoF-HLBra D228 D231 D253 D266 D267 D348 D385 D388 D405 D406 D411 D426 D427 D429 D840 Olsen Kt5 1 Potato Idaho 2012 25.4 27.5 28.1 27.4 28.4 27.7 - 27.9 27.3 28.9 29.1 27.8 27.9 27.8 28.4 26.9 Olsen Kt2 1 Potato Idaho 2012 27.8 30.2 30.2 29.5 31.2 30.2 - 30.1 30.2 31.1 31.2 29.7 30.1 30.1 31.3 29 ZC675 1 Potato Nebraska 2014 22 22.8 23.3 23 22.9 23.1 23.7 22.9 22.7 23.5 23.2 23.4 22.8 22.9 NT 28.2 ZC675-5 1 Potato Nebraska 2014 24.7 25.6 26.4 25.9 26.1 26.2 26.7 25.6 25.7 27.2 26.3 26.3 26 25.8 25.6 25.2 ID258-2 1 Potato Texas 2008 25.8 28.1 28.7 27.4 28.8 28.1 - 27.7 27.4 29.1 28.9 28.4 28.5 28.1 28.2 27 450T15 1 Potato Texas 2011 23.4 24.6 25.3 24.6 25.1 24.6 25.5 24.5 24.3 26.5 25.5 25.1 24.6 24.6 24.6 - ZC369 1 Potato Texas 2010 27.8 29.7 30.6 29.7 31.1 29.5 30.4 29.4 28.9 30.9 29.9 29.5 29.5 30.2 29.3 28.7 WA471-3 1 Potato Washington 2011 21.8 24 24.1 23.4 24.5 23.6 23.8 23.3 23.2 25.7 24.8 23.5 23.8 23.4 24.1 22.5 Tunnel2 1 Psyllids Guatemala 2013 24.2 27.4 27 26.8 28.3 26.5 27.7 26.1 26.1 28.9 28.5 26.3 27.4 26.1 28 25.1 HenB2 1 Psyllids Texas 2013 15.1 17.3 17.1 16.8 18.1 17.2 17.9 16.6 16.7 18.4 17.9 17.2 17.3 17 17.4 16.2 HenneB 1 Psyllids Texas 2013 20.2 22.3 22 24.3 30.3 22.3 27.3 21.3 22.7 - - 20.4 29.3 21.1 24.8 21.2 EggRootSe1 1,2 Eggplant Guatemala 2013 17.2 20 20.5 19.7 20.6 19.9 21.2 19.4 19.2 22.6 21.3 19.6 20.1 19.3 19.7 19 EggRootSe3 1,2 Eggplant Guatemala 2013 17.1 19.7 20 19.5 20.4 19.6 20.4 19.4 19.1 21.9 20.6 19.6 19.7 19.2 19.6 18.8 Olsen817 1,2 Potato Idaho 2013 24.2 26.5 27 26.2 27 26.4 26.8 26 26 27.4 27 26.5 27.7 26.3 27.1 25.5 Olsen859 1,2 Potato Idaho 2013 24.7 26.8 27.1 26.8 27.2 26.8 27.4 26.7 26.2 28 27.6 26.6 26.7 26.5 26.8 25.8 ZC457T1 1,2 Potato Washington 2011 26.6 32.7 - 33.1 31.4 32.3 34 32.1 32.3 34.1 - 33.7 32.2 31.1 33.4 32.2 TunnelVV 1,2 Psyllids Guatemala 2013 18.2 21 20.6 20.5 21.5 20.4 21.4 20 19.9 22.1 21.7 20.1 20.7 20.2 21.2 19.1 PO 1,2 Psyllids Texas 2013 16.6 18.3 18.5 18.3 18.3 18.5 19.1 18.4 18.1 18.5 18.4 18.8 18.1 18.1 17.8 18.1 HenF 1,2 Psyllids Texas 2013 13.9 26.8 26.6 26.1 26.9 26.2 18.8 26.1 25.7 27.7 27.6 26 26.4 20.4 26.8 25.2 HenL 1,2 Psyllids Texas 2014 17.8 19 19 NT 18.5 19 19.6 18.7 19.3 18.8 19.1 19.3 NT 18.6 NT 18.4 ZC398-2 2 Potato Colorado 2010 25.6 - - - * - - - - - - - - - - - FM7wSto 2 Potato Colorado 2012 21.2 - - - * - - * - * - - - 25.9 - - ZC673-14 2 Potato Colorado 2014 23.8 - - - - - - - - - - - - 29.3 - - ZC673-12 2 Potato Colorado 2014 23.8 - - - - - - - - - - - - 29.3 - - ZC673-7 2 Potato Colorado 2014 23.3 - - - - - - - - - - * - 28.9 - * TX418Sto2 2 Potato Texas 2011 19.4 - - - * - - - 29.4 - - - - 25.4 * - ZC674-2 2 Potato Texas 2014 25.2 - - - - - - - - - - - - 31.8 - - ZC672-5 2 Potato Texas 2014 26.8 - - - - - - - - - - - - 31 - - ZC431T8 2 Potato Texas 2011 22.5 - - - * - - - - - - * - 27.4 - - ZC429T3b 2 Potato Texas 2011 19.5 - - - - - - - - - - * - 25.8 - - ZC367-adj1 2 Potato Texas 2010 22 - - - - - - - * - - - - 32.6 * - ZC416T4b 2 Potato Texas 2011 24.2 - - - * - - - - - - - - 29.4 - - ZC414T1 2 Potato Texas 2011 24.8 - * - * - - - - - - - - 31 - - ZC421 T7 2 Potato Texas 2011 25.7 - - - * - - - - - - - - 32 - - ZC456T1 2 Potato Washington 2011 24.6 - - - * * - - - - - - - - - - Herm 2 Psyllids Oregon 2012 13.7 * - * * * 19.9 - * - * * * 20 - - HenneA1 2 Psyllids Texas 2013 26.5 - - - * - - - - - - - - - - - Edbg3 2 Psyllids Texas 2011 17.3 - - * - - - * - - - - - 23.6 - - 551Bulk 2 Psyllids Texas 2012 16.4 - - - * - - - - - - - - 23 - - 572C 2 Psyllids Texas 2012 16.1 - - - - - - - - - - - - 22.3 - - ZC572b 2 Psyllids Texas 2012 17.7 - - - - - - * - - - - - 23.6 - - HenW 2 Psyllids Texas 2012 25.3 - - - - - - - - - - - - - - - HenT 2 Psyllids Texas 2012 19.1 - - - * - 24.8 - - - - - - 24.6 - - A-B loci differentiation Within-haplotype A loci diversity (plus/ minus) Amplicons from haplotype B samples Non-specific amplicons Haplotype A target screening
- 9. biosecurity built on science Table 3. Cycle threshold values for screen of 15 qPCR assays against 43 samples Thompson, S. M., Johnson, C. P., Lu, A. Y., Frampton, R. A., Sullivan, K. L., Fiers, M.W. E. J., Crowhurst, R. N., Pitman, A. R., Scott, I. A.W., Wen, A., Gudmestad, N. C., and Smith, G. R. 2015. Genomes of ‘Candidatus Liberibacter solanacearum’ haplotype A from New Zealand and the United States suggest significant genome plasticity in the species. Phytopathology 105:863-871. Sample Clade Host Collected Year LsoF-HLBra D228 D231 D253 D266 D267 D348 D385 D388 D405 D406 D411 D426 D427 D429 D840 Olsen Kt5 1 Potato Idaho 2012 25.4 27.5 28.1 27.4 28.4 27.7 - 27.9 27.3 28.9 29.1 27.8 27.9 27.8 28.4 26.9 Olsen Kt2 1 Potato Idaho 2012 27.8 30.2 30.2 29.5 31.2 30.2 - 30.1 30.2 31.1 31.2 29.7 30.1 30.1 31.3 29 ZC675 1 Potato Nebraska 2014 22 22.8 23.3 23 22.9 23.1 23.7 22.9 22.7 23.5 23.2 23.4 22.8 22.9 NT 28.2 ZC675-5 1 Potato Nebraska 2014 24.7 25.6 26.4 25.9 26.1 26.2 26.7 25.6 25.7 27.2 26.3 26.3 26 25.8 25.6 25.2 ID258-2 1 Potato Texas 2008 25.8 28.1 28.7 27.4 28.8 28.1 - 27.7 27.4 29.1 28.9 28.4 28.5 28.1 28.2 27 450T15 1 Potato Texas 2011 23.4 24.6 25.3 24.6 25.1 24.6 25.5 24.5 24.3 26.5 25.5 25.1 24.6 24.6 24.6 - ZC369 1 Potato Texas 2010 27.8 29.7 30.6 29.7 31.1 29.5 30.4 29.4 28.9 30.9 29.9 29.5 29.5 30.2 29.3 28.7 WA471-3 1 Potato Washington 2011 21.8 24 24.1 23.4 24.5 23.6 23.8 23.3 23.2 25.7 24.8 23.5 23.8 23.4 24.1 22.5 Tunnel2 1 Psyllids Guatemala 2013 24.2 27.4 27 26.8 28.3 26.5 27.7 26.1 26.1 28.9 28.5 26.3 27.4 26.1 28 25.1 HenB2 1 Psyllids Texas 2013 15.1 17.3 17.1 16.8 18.1 17.2 17.9 16.6 16.7 18.4 17.9 17.2 17.3 17 17.4 16.2 HenneB 1 Psyllids Texas 2013 20.2 22.3 22 24.3 30.3 22.3 27.3 21.3 22.7 - - 20.4 29.3 21.1 24.8 21.2 EggRootSe1 1,2 Eggplant Guatemala 2013 17.2 20 20.5 19.7 20.6 19.9 21.2 19.4 19.2 22.6 21.3 19.6 20.1 19.3 19.7 19 EggRootSe3 1,2 Eggplant Guatemala 2013 17.1 19.7 20 19.5 20.4 19.6 20.4 19.4 19.1 21.9 20.6 19.6 19.7 19.2 19.6 18.8 Olsen817 1,2 Potato Idaho 2013 24.2 26.5 27 26.2 27 26.4 26.8 26 26 27.4 27 26.5 27.7 26.3 27.1 25.5 Olsen859 1,2 Potato Idaho 2013 24.7 26.8 27.1 26.8 27.2 26.8 27.4 26.7 26.2 28 27.6 26.6 26.7 26.5 26.8 25.8 ZC457T1 1,2 Potato Washington 2011 26.6 32.7 - 33.1 31.4 32.3 34 32.1 32.3 34.1 - 33.7 32.2 31.1 33.4 32.2 TunnelVV 1,2 Psyllids Guatemala 2013 18.2 21 20.6 20.5 21.5 20.4 21.4 20 19.9 22.1 21.7 20.1 20.7 20.2 21.2 19.1 PO 1,2 Psyllids Texas 2013 16.6 18.3 18.5 18.3 18.3 18.5 19.1 18.4 18.1 18.5 18.4 18.8 18.1 18.1 17.8 18.1 HenF 1,2 Psyllids Texas 2013 13.9 26.8 26.6 26.1 26.9 26.2 18.8 26.1 25.7 27.7 27.6 26 26.4 20.4 26.8 25.2 HenL 1,2 Psyllids Texas 2014 17.8 19 19 NT 18.5 19 19.6 18.7 19.3 18.8 19.1 19.3 NT 18.6 NT 18.4 ZC398-2 2 Potato Colorado 2010 25.6 - - - * - - - - - - - - - - - FM7wSto 2 Potato Colorado 2012 21.2 - - - * - - * - * - - - 25.9 - - ZC673-14 2 Potato Colorado 2014 23.8 - - - - - - - - - - - - 29.3 - - ZC673-12 2 Potato Colorado 2014 23.8 - - - - - - - - - - - - 29.3 - - ZC673-7 2 Potato Colorado 2014 23.3 - - - - - - - - - - * - 28.9 - * TX418Sto2 2 Potato Texas 2011 19.4 - - - * - - - 29.4 - - - - 25.4 * - ZC674-2 2 Potato Texas 2014 25.2 - - - - - - - - - - - - 31.8 - - ZC672-5 2 Potato Texas 2014 26.8 - - - - - - - - - - - - 31 - - ZC431T8 2 Potato Texas 2011 22.5 - - - * - - - - - - * - 27.4 - - ZC429T3b 2 Potato Texas 2011 19.5 - - - - - - - - - - * - 25.8 - - ZC367-adj1 2 Potato Texas 2010 22 - - - - - - - * - - - - 32.6 * - ZC416T4b 2 Potato Texas 2011 24.2 - - - * - - - - - - - - 29.4 - - ZC414T1 2 Potato Texas 2011 24.8 - * - * - - - - - - - - 31 - - ZC421 T7 2 Potato Texas 2011 25.7 - - - * - - - - - - - - 32 - - ZC456T1 2 Potato Washington 2011 24.6 - - - * * - - - - - - - - - - Herm 2 Psyllids Oregon 2012 13.7 * - * * * 19.9 - * - * * * 20 - - HenneA1 2 Psyllids Texas 2013 26.5 - - - * - - - - - - - - - - - Edbg3 2 Psyllids Texas 2011 17.3 - - * - - - * - - - - - 23.6 - - 551Bulk 2 Psyllids Texas 2012 16.4 - - - * - - - - - - - - 23 - - 572C 2 Psyllids Texas 2012 16.1 - - - - - - - - - - - - 22.3 - - ZC572b 2 Psyllids Texas 2012 17.7 - - - - - - * - - - - - 23.6 - - HenW 2 Psyllids Texas 2012 25.3 - - - - - - - - - - - - - - - HenT 2 Psyllids Texas 2012 19.1 - - - * - 24.8 - - - - - - 24.6 - - A-B loci differentiation Within-haplotype A loci diversity (plus/ minus) Amplicons from haplotype B samples Non-specific amplicons Haplotype A target screening Benefit to researchers and biosecurity diagnosticians: The ability to detect dual infections
- 10. biosecurity built on science Origin of the New Zealand CLso incursion 29 New Zealand samples as extracted DNA - From a range of hosts, times and locations - Positive by 16S rRNA qPCR assay (Beard et al 2013) Selected three loci (two variable, one consistent) - D348 (17/20 American A) - D406 (18/20 American A) - D426 (19/19 American A) Sample Sample Host Location Collected M8e TPP Thorn apple Hawke’s Bay 3-Apr-14 W15 TPP Jerusalem cherry Hawke’s Bay 6-Jun-14 W23#36 TPP Jerusalem cherry Hawke’s Bay 8-Oct-14 tpp+n TPP (nymph) Boxthorn Canterbury 30-May-14 806 AB 2 TPP Boxthorn Hawke’s Bay 29-Feb-12 876 A8 TPP Boxthorn Canterbury 21-Sep-12 875 A2 TPP Boxthorn Canterbury 21-Sep-12 M15a TPP Potato Hawke’s Bay 3-Apr-14 M15D TPP Potato Hawke’s Bay 3-Apr-14 HBL1 TPP unknown Hawke’s Bay unknown MHBN 6 TPP unknown Hawke’s Bay unknown FMAN B8 TPP Potato Manawatu unknown FMAN A3 TPP Potato Manawatu unknown M RAK 7 TPP Potato Canterbury 4-Apr-12 F RAK 2 TPP Potato Canterbury 4-Apr-12 MWAI 9 TPP unknown Auckland unknown 343 Plant Potato Auckland unknown JA Pot+ Plant Potato Auckland 1-Jul-09 M7 Plant Potato Canterbury 20-Mar-14 M4 Plant Potato Canterbury 20-Mar-14 M6 Plant Potato Canterbury 20-Mar-14 A7 tom Plant Tomato Marlborough Jan-12 Is5 Plant Tomato Marlborough unknown Is4 Plant Tomato Marlborough unknown Wat Tom 1 Plant Tomato unknown unknown FZ 3B2 Plant Tomato Marlborough unknown TAM3 Plant Tamarillo Northland unknown tpp+ (C22) TPP (colony) Potato Auckland 2011 tpp- (C20) TPP (colony) Tomato Auckland 2006
- 11. biosecurity built on science Origin of the New Zealand CLso incursion 29 New Zealand samples as extracted DNA - From a range of hosts, times and locations - Positive by 16S rRNA qPCR assay (Beard et al 2013) Selected three loci (two variable, one consistent) - D348 (17/20 American A) - D406 (18/20 American A) - D426 (19/19 American A) Sample Sample Host Location Collected M8e TPP Thorn apple Hawke’s Bay 3-Apr-14 W15 TPP Jerusalem cherry Hawke’s Bay 6-Jun-14 W23#36 TPP Jerusalem cherry Hawke’s Bay 8-Oct-14 tpp+n TPP (nymph) Boxthorn Canterbury 30-May-14 806 AB 2 TPP Boxthorn Hawke’s Bay 29-Feb-12 876 A8 TPP Boxthorn Canterbury 21-Sep-12 875 A2 TPP Boxthorn Canterbury 21-Sep-12 M15a TPP Potato Hawke’s Bay 3-Apr-14 M15D TPP Potato Hawke’s Bay 3-Apr-14 HBL1 TPP unknown Hawke’s Bay unknown MHBN 6 TPP unknown Hawke’s Bay unknown FMAN B8 TPP Potato Manawatu unknown FMAN A3 TPP Potato Manawatu unknown M RAK 7 TPP Potato Canterbury 4-Apr-12 F RAK 2 TPP Potato Canterbury 4-Apr-12 MWAI 9 TPP unknown Auckland unknown 343 Plant Potato Auckland unknown JA Pot+ Plant Potato Auckland 1-Jul-09 M7 Plant Potato Canterbury 20-Mar-14 M4 Plant Potato Canterbury 20-Mar-14 M6 Plant Potato Canterbury 20-Mar-14 A7 tom Plant Tomato Marlborough Jan-12 Is5 Plant Tomato Marlborough unknown Is4 Plant Tomato Marlborough unknown Wat Tom 1 Plant Tomato unknown unknown FZ 3B2 Plant Tomato Marlborough unknown TAM3 Plant Tamarillo Northland unknown tpp+ (C22) TPP (colony) Potato Auckland 2011 tpp- (C20) TPP (colony) Tomato Auckland 2006 Benefit to researchers and in incursion response: tools for assessing genetic diversity within a haplotype
- 12. biosecurity built on science Industry submission to MPI Risk Assessment of CLso B Provide information for Market Access Solutionz submission to MPI on implications of CLso B for potato, tomato and capsicum industries 1. CLso B does appear to be more pathogenic to both plants and psyllids than CLso A 2. There are significant genetic differences between these two haplotypes (genome organisation, unique genes, prophage sequences) 3. There is genetic variation within both the A and B haplotypes in the USA, evidenced by presence/ absence of putative haplotype differential diagnostic loci (genes) 4. New Zealand (and Norfolk Island) appear to have extremely limited genetic diversity of CLso A, based on the research we have undertaken to assess diversity 5. There are three other CLso haplotypes (C D E) that we know of. 6. There are at least four biotypes of TPP described in the USA. New Zealand (and Norfolk Island) only have one of the biotypes, so keeping these other variants out is also important
- 13. biosecurity built on science Industry submission to MPI Risk Assessment of CLso B Provide information for Market Access Solutionz submission to MPI on implications of CLso B for potato, tomato and capsicum industries 1. CLso B does appear to be more pathogenic to both plants and psyllids than CLso A 2. There are significant genetic differences between these two haplotypes (genome organisation, unique genes, prophage sequences) 3. There is genetic variation within both the A and B haplotypes in the USA, evidenced by presence/ absence of putative haplotype differential diagnostic loci (genes) 4. New Zealand (and Norfolk Island) appear to have extremely limited genetic diversity of CLso A, based on the research we have undertaken to assess diversity 5. There are three other CLso haplotypes (C D E) that we know of. 6. There are at least four biotypes of TPP described in the USA. New Zealand (and Norfolk Island) only have one of the biotypes, so keeping these other variants out is also important Benefit to end-user: MPI Understanding of the taxonomy contributing to informed regulatory decisions
- 14. biosecurity built on science Summary • Genome assemblies including the first haplotype A assembly • Tools for assessing the within haplotype diversity • qPCR assays for differentiating the solanaceous-infecting haplotypes • Provide science input to inform the decision around deregulating haplotype B in NZ
- 15. biosecurity built on science End-User’s Perspective “The team have clearly established the value of bioinformatic tools to identify diagnostic targets in an unculturable bacterium. In doing so they have revealed a level of genomic plasticity and complexity in a pathogen only first identified in 2008. The identification protocol the team will recommend via a National Diagnostic Protocol will have a substantial amount of information and critical analysis supporting the new diagnostic”. - Barbara Hall Chair of SPHD
- 16. biosecurity built on science Benefit to industry
- 17. biosecurity built on science Future work Psyllid mitochondrial genomes - ~200 genomes at various stages of assembly - Metagenomic not PCR assembly - Diversity, origin Access to new CLso samples (eg Honduran) - Diversity, origin - Improved diagnostics Technology Sequencing options (DNA and RNA) Assembly strategies Diagnostic systems Looking forward Finalise/ conclude ~40 CLxx assemblies Validate the diagnostic assays Finalise international diagnostic protocols Basis of CLso pathogenicity CLso targets for control options
Editor's Notes
- This work was carried out under project CRC2002 and 2156 Genome-Informed diagnostics for bacterial pathovars. Our culprit of choice is Candidatus Liberibacter solanacearum. The story begins in the mid-90s when a disease in potatoes first started appearing in the US where potato chips were presenting with darkened arrays or stripes giving the disease its name zebra chip disease.
- The putative causal agent Liberibacter solanacearum was described in 2008. Its an alpha proteo bacteria that is vectored by psyllids – phloem feeding insects. There are 5 haplotypes of Lso reported - A through to E – defined based on differences in the ribosomal RNA operon.
- The 5 haplotypes are associated with different hosts. A and B are found on solanaceous plants, C is reported on carrots and and D and E are on carrots and celery.
- They are also found in different geographical ranges. A and B are found central and North America and are vectored by Bactericera cockerelli (TPP). A is also found in New Zealand and the Australian territory of Norfolk Island. C is in Scandinavia and vectored by Trioza apicalis while is D and E are in Europe and North Africa and their main vector appears to be Bactericera trigonica.
- At present Lso is not reported on Mainland Australia. It has been identified as a major threat to the Australian potato industry and is considered by many to be its no1 biosecurity threat. It is on Plant Health Australia’s list of High Priority Pests. Jim did a good job of highlighting the importance of understanding what the names we use in biosecurity represent. At the start of this project little was known about the Lso haplotypes beyond the rRNA sequence and the only way to identify the haplotype is through sequencing part of it. Aside from the time factor this technique does not detect dual infections which there is growing evidence for. I mentioned previously that Lso is the putative causal agent of zebra chip disease. We describe it as putative because it is unculturable: hence its classification in the family Candidatus. This means that we don’t have pure cultures of it and instead have used sample of the bacteria in host for sequencing. Then have to assemble the genomes from a background of host and endosymbiont sequence. Future research: biological differences between haplotypes?
- While sequenced Lso from samples from various European countries, North America, Central America and New Zealand here I have taken just a representative sequence for each of 4 haplotypes to summarise the results. These are the results of the average nucleotide identity analysis. This is a pairwise comparison where a genome is chopped into sections and each section is aligned to a second genome and the percentage identity of the alignment is calculated. The average percentage identity is then calculated giving the ANI. A threshold of above 95% is generally considered to be the same species. This type of analysis is used to give an indication of the evolutionary history of the genus/species. Here you can see that haplotype A has 98,29% and 97.77% to the apiaceous-infecting haplotypes D and E while its identity to haplotype B is lower at 97.69%! This is despite infecting the same hosts in the same geographic locations as haplotype B. This suggests that they have had either a different host or geographical range at some point in history. It raises questions around where they resided and in what prior to Lso emerging as a major potato disease in the last 20 years. Understanding what drives these changes and how they occur can help us identify future biosecurity threats
- Following genome sequencing comparison of all the genomes allowed us to identify regions that were only found in the the solanaceous-infecting haplotypes – either A or B. We used these regions to target for haplotype specific qPCR assays. In the interests of time here I have just presented the screening results for the haplotypeA targets against a panel of samples from around North and Central America. Some assays amplified products from hap B samples (in red) and some assays did not amplify all hap Atargets. This indicates that these loci are not fixed within the haplotype – its possible that some of these loci are or were on mobile genetic elements. It demonstrates that there is significant genetic diversity within the haplotypes in the Americas. However the ones in green did amplify all hap A targets and no hap B targets. These targets were subsequently subjected to extensive screening against a series of panels including other liberibacters, other microbes that infect solanaceous crops, healthy hosts and so forth.
- How many people genome sequence their isolate these days for a biological experiment? How many people know what HGT/phage elements their organism has? So often genomic studies are criticised for not having biology but the it’s a two-way street – biological studies will increasingly need genomic data to characterise their work. Does this also apply to viral, fungal and insect studies?
- While not all of the targets proved fixed across haplotype A samples they have proved useful for looking at genetic diversity. To assess the genetic diversity of the Lso in NZ 3 of the assays were chosen – 2 with variable regions and one fixed. These were tested against a panel of 29 DNA samples from a range of hosts, times and collection locations. All samples amplified with all 3 targets indicating that NZ has less genetic diversity than the states. This is consistent with a recent, limited incursion. They were also tested against samples from Norfolk Island. The Norfolk Is samples gave the same profiles as the NZ samples supporting the theory that the liberibacter in NI came from NZ.
- While not all of the targets proved fixed across haplotype A samples they have proved useful for looking at genetic diversity. To assess the genetic diversity of the Lso in NZ 3 of the assays were chosen – 2 with variable regions and one fixed. These were tested against a panel of 29 DNA samples from a range of hosts, times and collection locations. All samples amplified with all 3 targets indicating that NZ has less genetic diversity than the states. This is consistent with a recent, limited incursion. They were also tested against samples from Norfolk Island. The Norfolk Is samples gave the same profiles as the NZ samples supporting the theory that the liberibacter in NI came from NZ.
- At present MPI is undertaking a risk assessment of Clso B to investigate deregulating it as a biosecurity threat to NZ. Based on our research we have provided information for the industry submission. Given the substantial differences between haplotypes A and B and the genetic diversity within haplotypes we recommend that haplotype B remains a biosecurity threat as any additional genetic diversity in NZ could be problematic.
- At present MPI is undertaking a risk assessment of Clso B. Based on
- Subcommitte for Plant Health Diagnostics
- Continuing research – field diagnostics – Rebekah’s talk Understanding the mechanisms driving plant response/disease expression Review: from genomics to application…