Session 2: Liberibacter solanacearum comparative genomics and diagnostics

biosecurity built on science

Liberibacter solanacearum comparative genomics and diagnostics

Creswick, Science Exchange, 26-28th Oct 2016Sarah Thompson, Chris Johnson, Rebekah Frampton, Kerry Sullivan,

Charles David, Falk Kalamorz, Neil Gudmestad, Grant Smith.


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


Liberibacter solanacearum haplotypes

A and B

C

D and E


Liberibacter solanacearum haplotypes

A

A and B

C

D and E


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)


ANI comparison of the draft genomes

Genus Species SubspeciesANI 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


ANI comparison of the draft genomes

Genus Species SubspeciesANI 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


Table 3. Cycle threshold values for screen of 15 qPCR assays against 43 samplesThompson, 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 D840Olsen 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 26.9Olsen 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 29ZC675 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 28.2ZC675-5 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 25.2ID258-2 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 27450T15 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 28.7WA471-3 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 22.5Tunnel2 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 25.1HenB2 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 16.2HenneB 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 21.2EggRootSe1 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 19EggRootSe3 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 18.8Olsen817 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 25.5Olsen859 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 25.8ZC457T1 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 32.2TunnelVV 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 19.1PO 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 18.1HenF 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 25.2HenL 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 18.4ZC398-2 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


Table 3. Cycle threshold values for screen of 15 qPCR assays against 43 samplesThompson, 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 D840Olsen 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 26.9Olsen 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 29ZC675 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 28.2ZC675-5 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 25.2ID258-2 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 27450T15 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 28.7WA471-3 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 22.5Tunnel2 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 25.1HenB2 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 16.2HenneB 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 21.2EggRootSe1 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 19EggRootSe3 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 18.8Olsen817 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 25.5Olsen859 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 25.8ZC457T1 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 32.2TunnelVV 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 19.1PO 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 18.1HenF 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 25.2HenL 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 18.4ZC398-2 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


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 CollectedM8e TPP Thorn apple Hawke’s Bay 3-Apr-14W15 TPP Jerusalem cherry Hawke’s Bay 6-Jun-14

W23#36 TPP Jerusalem cherry Hawke’s Bay 8-Oct-14tpp+n TPP (nymph) Boxthorn Canterbury 30-May-14

806 AB 2 TPP Boxthorn Hawke’s Bay 29-Feb-12876 A8 TPP Boxthorn Canterbury 21-Sep-12875 A2 TPP Boxthorn Canterbury 21-Sep-12M15a TPP Potato Hawke’s Bay 3-Apr-14M15D TPP Potato Hawke’s Bay 3-Apr-14HBL1 TPP unknown Hawke’s Bay unknown

MHBN 6 TPP unknown Hawke’s Bay unknownFMAN B8 TPP Potato Manawatu unknownFMAN A3 TPP Potato Manawatu unknownM RAK 7 TPP Potato Canterbury 4-Apr-12F RAK 2 TPP Potato Canterbury 4-Apr-12MWAI 9 TPP unknown Auckland unknown

343 Plant Potato Auckland unknownJA Pot+ Plant Potato Auckland 1-Jul-09

M7 Plant Potato Canterbury 20-Mar-14M4 Plant Potato Canterbury 20-Mar-14M6 Plant Potato Canterbury 20-Mar-14

A7 tom Plant Tomato Marlborough Jan-12Is5 Plant Tomato Marlborough unknownIs4 Plant Tomato Marlborough unknown

Wat Tom 1 Plant Tomato unknown unknownFZ 3B2 Plant Tomato Marlborough unknownTAM3 Plant Tamarillo Northland unknown

tpp+ (C22) TPP (colony) Potato Auckland 2011tpp- (C20) TPP (colony) Tomato Auckland 2006


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 CollectedM8e TPP Thorn apple Hawke’s Bay 3-Apr-14W15 TPP Jerusalem cherry Hawke’s Bay 6-Jun-14

W23#36 TPP Jerusalem cherry Hawke’s Bay 8-Oct-14tpp+n TPP (nymph) Boxthorn Canterbury 30-May-14

806 AB 2 TPP Boxthorn Hawke’s Bay 29-Feb-12876 A8 TPP Boxthorn Canterbury 21-Sep-12875 A2 TPP Boxthorn Canterbury 21-Sep-12M15a TPP Potato Hawke’s Bay 3-Apr-14M15D TPP Potato Hawke’s Bay 3-Apr-14HBL1 TPP unknown Hawke’s Bay unknown

MHBN 6 TPP unknown Hawke’s Bay unknownFMAN B8 TPP Potato Manawatu unknownFMAN A3 TPP Potato Manawatu unknownM RAK 7 TPP Potato Canterbury 4-Apr-12F RAK 2 TPP Potato Canterbury 4-Apr-12MWAI 9 TPP unknown Auckland unknown

343 Plant Potato Auckland unknownJA Pot+ Plant Potato Auckland 1-Jul-09

M7 Plant Potato Canterbury 20-Mar-14M4 Plant Potato Canterbury 20-Mar-14M6 Plant Potato Canterbury 20-Mar-14

A7 tom Plant Tomato Marlborough Jan-12Is5 Plant Tomato Marlborough unknownIs4 Plant Tomato Marlborough unknown

Wat Tom 1 Plant Tomato unknown unknownFZ 3B2 Plant Tomato Marlborough unknownTAM3 Plant Tamarillo Northland unknown

tpp+ (C22) TPP (colony) Potato Auckland 2011tpp- (C20) TPP (colony) Tomato Auckland 2006

Benefit to researchers and in incursion response: tools for assessing genetic diversity within a haplotype


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 A2. 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 diversity5. 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


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 A2. 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 diversity5. 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: MPIUnderstanding of the taxonomy contributing to informed regulatory

decisions


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


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


Benefit to industry


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

Microflora- Carsonella and other

endosymbiont genomes- Diversity, origin

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