insert presentation title...comparative liberibacter genomics comparative genomics of the...

biosecurity built on science

Jacqueline Morris

PhD Candidate

Plant Biosecurity Cooperative Research Centre

‘Candidatus Liberibacter

brunswickensis’ identified in the

Australian eggplant psyllid


Psyllid microflora

▪ Dependent on their microflora for essential nutrients

▪ Some species are known to vector disease-causing microbes - Candidatus Liberibacter solanacearum- Candidatus Liberibacter asiaticus

▪ No Liberibacter species have been detected in mainland Australia

▪ Diagnostics tests for the Liberibacter species have not been validated in Australia

Tomato potato psyllidThe New Zealand Institute for Plant and Food Research Limited.

http://amarillo.tamu.edu/about-our-website-2/diseases-of-

potato/zebra-chip-description/symptom-gallery/

Asian citrus psyllid

http://www.sandiegocounty.gov/awm/acp.html

Huanglongbing affected

orange and leaves

http://www.livescience.com/30050-citrus-greening-

destroy-orange-crop.html

Zebra chip affected tuber

Global distribution of the Liberibacter genus.

Adapted from Haapalainen et al., 2014


Native Australian Psyllids

▪ Australia is a centre of psyllid diversity

▪ Little is known about the microflora of native Australian psyllids- Could the native microflora confound diagnostic tests for phytopathogenic Ca. Liberibacter species?- Are there Ca. Liberibacter species in native Australian psyllids?

▪ Understanding the microflora of Australian psyllids is important for both biosecurity preparedness and response management of exotic diseases

Estimated 446 species present (Yen, 2002)


Acizzia solanicola

Acizzia solanicola

Photo: Linda Samenaro

▪ Acizzia solanicola (Hemiptera: Psyllidae), eggplant psyllid

▪ Broadened host range

▪ Host range cross over with the tomato potato psyllid-which has recently been detected in Western Australia

Current known hosts and distribution of A. solanicola in Australia, adapted from Kent & Taylor, 2010; Taylor & Kent, 2013

Known hosts for A. solanicola

Solanum pterophilum Rock nightshade

Solanum melongena Eggplant

Solanum mauritianum Wild tobacco bush

Physalis peruviana Cape gooseberry

Brugmansia Undetermined species of angel’s trumpet

biosecurity built on sciencePhylogenetic tree of cloned 16S rRNA region (1403 bp) Morris et al., 2017

▪ Designed generic Liberibacter genus primers based on the 16S rRNA region to detect all known liberibacter species

Liberibacter-like microflora?

Close

relatives

Liberibacters


▪ Most liberibacters are unculturable, therefore need to sequence the metagenome (psyllid and associated microflora)

▪ Perform Illumina sequencing (Miseq and NextSeq)

▪ De novo assembly of a novel bacterial genome from a metagenomic dataset is difficult

- Liberibacter genomes contain long repeat regions, varying prophages, have a low GC content

▪ No reference genome

▪ Multi locus sequence analyses (MLSA)

Metagenomic sequencing

= ~20 million reads

(20,868,677)

= ~1 billion reads(1,182,234,928)

x 50MiSeq NextSeq


▪ Two sets of 7 genes that were 100% identical to their individual analyses A. solanicola-associated Ca. Liberibacter species and CLaf

▪ No chimeras formed- spiking the dataset proved that the closest relative, did not change the contigs formed

Multi locus sequence analyses (MLSA) mapping approach

Morris et al., 2017


MLSA phylogeny (12,617 bp)

▪ Candidatus Liberibacter brunswickensis (CLbr)

Disease associated

No disease associated

Alpha Proteobacteria relatives

Outgroup


Implications for diagnostics

▪ There is microflora in native Australian psyllids that can confound diagnostic tests developed outside of Australia

▪ Important to ensure diagnostic tests are validated within each region

CLaf

Li et al., 2006

CLas

Li et al., 2006

CLam

Li et al., 2006

CLso

Li et al., 2009

CLaf + - - -

CLas - + - -

CLam - - + -

CLso - - - +

CLbr + + - -

qPCR protocols recommended in the NDP (Australia), EPPO and IPPC


A. solanicola metagenomic dataset

▪ Improve assembly of the CLbr sequence data- Illumina MiSeq- Illumina NextSeq

▪ Map to all Liberibacters, perform an assembly and assess the contigs

Metagenomic dataset benefits

▪ A. solanicola mitochondrial genome

▪ Primary endosymbionts- Candidatus Carsonella


Comparative Liberibacter genomics

▪ Comparative genomics of the Liberibacter genus with New Zealand Plant and Food Research (PBCRC2156- Bacterial pathovars)

▪ Largest comparison of Liberibacter dataset including novel species

- 37 genomes (complete and draft)

- Including 7 species

▪ Genome features for species and genus

- Core

- Pan

- Accessory Fagen et al., 2014


▪ CLbr has been detected in eggplants of a large colony

- Leaf tissue, midribs, petiole, stems and roots

▪ Experiments designed

- To determine if A. solanicola can acquire CLbr from an eggplant

- Compare the feeding of A. solanciola with and without CLbr

- Identify the location of CLbr and the primary endosymbiont Ca. Carsonella in A. solanicola(Fluorescent in situ hybridisation)

Association of CLbr, psyllid and plant

http://www.edmontonjournal.com/plan+early+peppers+

eggplants/7849981/story.html

Ghanim et al., 2016


Summary

▪ A new species of Ca. Liberibacter has been detected in A. solanicola

▪ Plant disease associated with the presence of the bacteria has not been observed

▪ Candidatus Liberibacter brunswickensis (CLbr)

▪ This is the first time a Ca. Liberibacter species has been detected in mainland Australia and from the psyllid genus Acizzia

▪ Future work into the biology and genomics of CLbr


Thank you!

PBCRC Bacterial Pathovars (PBCRC2156)

▪ Rebekah Frampton

▪ Sarah Thompson

▪ Falk Kalamorz

▪ The Plant Micro Group

▪ Debbie Kent

▪ Isabel Valenzuela-Gonzalez

▪ Piotr Trebicki

▪ Jessica Dohmen-Vereijssen

Project team (PBCRC 62116)

▪ Brendan Rodoni

▪ Rachel Mann

▪ Jason Shiller

▪ Grant Smith

▪ Malik Malipatil

▪ Alan Yen

insert presentation title...comparative liberibacter genomics comparative genomics of the...

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