biosecurity built on science
Jacqueline Morris
PhD Candidate
Plant Biosecurity Cooperative Research Centre
‘Candidatus Liberibacter
brunswickensis’ identified in the
Australian eggplant psyllid
biosecurity built on science
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
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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)
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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
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▪ 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
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▪ 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
biosecurity built on science
MLSA phylogeny (12,617 bp)
▪ Candidatus Liberibacter brunswickensis (CLbr)
Disease associated
No disease associated
Alpha Proteobacteria relatives
Outgroup
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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
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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
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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
biosecurity built on science
▪ 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
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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
biosecurity built on science
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