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Jacqueline Morris
PhD Candidate
Plant Biosecurity Cooperative Research Centre
Psyllid microflora- Implications for liberibacter disease surveillance
and pest control
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Psyllid microflora
Dependent on their microflora for essential nutrients
Known to vector disease-causing microbes, two of which are;
- Candidatus Liberibacter solanacearum causes zebra chip in potatoes
- Candidatus Liberibacter asiaticus causes citrus greening in citrus
Recent detection of tomato potato psyllid in Western Australia
None of the pathogenic liberibacter species of concern have been detected in mainland Australia
Diagnostics tests for the liberibacter species have been developed outside of 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/
Global distribution of the liberibacter genus. Adapted from Haapalainen et al., 2014
Asian citrus psyllid
http://www.sandiegocounty.gov/awm/acp.html
Citrus greening affected
orange and leaves
http://www.livescience.com/30050-citrus-
greening-destroy-orange-crop.html
Zebra chip affected
tuber
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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. Liberibacterspecies?- Can native Australian psyllids vector or host phytopathogenic Ca. Liberibacter species?
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, eggplant psyllidPhoto: Linda Samenaro
Acizzia solanicola, a native Australian psyllid, commonly known as the eggplant psyllid selected as a starting point
Broadened host range from the native solanaceous host plant the rock nightshade, Solanum pterophilum, to eggplants, Solanum melongena wild tobacco bush, Solanum mauritianumcape gooseberry, Physalis peruvianaundetermined species of angel’s trumpet, Brugmansia(Kent & Taylor, 2010; Taylor & Kent, 2013)
Host range cross over with the tomato potato psyllid
Current known distribution of A. solanicola in
Australia, adapted from Taylor & Kent, 2013.
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Liberibacter-like microflora?
Designed generic liberibacter genus primers to detect all known liberibacter species (Morris et al.,2017)
Apply primers to A. solanicola individuals
17 out of 37 A. solanicola individuals generated amplicons of the correct size (684 bp)
All amplicon sequences were identical and fell within the liberibacter genus - 99% identity to CLso species
Cloned the full length 16S rRNA region (~ 1500 bp)
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16S rRNA region phylogeny (1403 bp)
Phylogenetic tree of cloned 16S rRNA region (1403 bp) of A. solanicola-associated Ca. Liberibacter species
Disease associated
No disease associated
Alpha Proteobacteria relatives
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Most liberibacters are unculturable, therefore need to sequence the metagenome (psyllid and associated microflora)
Perform a Miseq and NextSeq run with the same library
De novo assembly of bacterial genome from a metagenomic dataset is difficult
- Liberibacter genomes contain long repeat regions, varying prophages, low GC content
- No reference genome
Perform multi locus sequence analyses (MLSA)
Increase sequence data
= ~20 million reads
(20,868,677)
= ~1 billion reads(1,182,234,928)
x 50MiSeq NextSeq
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Multi locus sequence analyses (MLSA) mapping approach
7 highly conserved genes in the Liberibacter genus
CLas as a reference
Map the A. solanicola metagenome (psyllid and bacterial DNA)
Take all the reads that mapped
Perform a de novo assembly
7 genes for the A. solanicola-associated Ca. Liberibacter species
dnaG - gyrB - mutS - nusG - rplA - rpoB - tufB
A. solanicola-associated Ca. Liberibacter
dnaG - gyrB - mutS - nusG - rplA - rpoB - tufBCLas
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MLSA mapping approach
To help prove the MLSA did not create chimeric contigs
Closest known species = CLaf
Created simulated reads, by chopping up the genome
All 7 genes assembled for CLaf
CLaf
CLaf only
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Mix both datasets to spike and repeat
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
MLSA mapping approach
A. solanicola-associated Ca. Liberibacter
CLaf
Acizzia solanicola metagenome+ CLaf spiked
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MLSA phylogeny (12,617 bp)
Concatenate all 7 genes for each species
Disease associated
No disease associated
Alpha Proteobacteria relatives
Outgroup
Multi locus sequence phylogenetic tree (12,617 bp)
Candidatus Liberibacter brunswickensis
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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 Australia
Finally it is important that diagnostics tests are not based on a single gene, in particular the 16S rRNA region
CLafHLBafLi et al,. 2006
CLasHLBasLi et al,. 2006
CLamHLBamLi et al,. 2006
CLsoLsoFLi et al,. 2009
CLaf + - - -
CLas - + - -
CLam - - + -
CLso - - - +
CLbr + + - -
qPCR protocols recommended in the NDP, IPPC and EPPO
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Summary
A new species of Ca. Liberibacter has been detected in A. solanicola, a native Australian psyllid
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
Manuscript accepted February 2017
Novel ‘Candidatus Liberibacter’ species identified in the
Australian eggplant psyllid, Acizzia solanicolaJacqueline Morris 1,2,3, Jason Shiller 1,3,4, Rachel Mann1,3, Grant Smith1,5,6,
Alan Yen 1,2,3, Brendan Rodoni 1,2,3
1. Plant Biosecurity Cooperative Research Centre, LPO Box 5012, Bruce, Australian Capital Territory, 2617, Australia 2. La
Trobe University, AgriBio, 5 Ring Road, Bundoora, Victoria, 3083, Australia 3. Agriculture Victoria, AgriBio, 5 Ring Road,
Bundoora, Victoria , 3083, Australia 4. INRA / Université d'Angers - IRHS Batiment C, 42 rue Georges Morel, Beaucouzé,
49071, France 5. Plant & Food Research Lincoln, Gerald St, Lincoln, 7608, New Zealand 6. Better Border Biosecurity,
Lincoln, 7608, New Zealand
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Association of CLbr, psyllid and plant
Experiments designed
To determine the acquisition of CLbr from eggplants by A. solanicola
Perform inoculation experiments and 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)
CLbr
+ve
CLbr
-ve
No
psyllids
So far
Large colony feeding on eggplants for over 2 months
CLbr has been detected in eggplants of this colony- Leaf tissue, midribs, stems and roots http://www.edmontonjournal.com/plan+ear
ly+peppers+eggplants/7849981/story.html
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Genomics
Improve assembly of the CLbr sequence data- Illumina NextSeq and PacBio datasets
Comparative genomics of the liberibacter genus with New Zealand Plant and Food Research (PBCRC2156- Bacterial pathovars)
Large dataset including most species of liberibacter - What are the core, pan and accessory genes?
- Can the comparative genomics analysis help us understand liberibacters further?
- Can the comparative genomic analysis help inform diagnostics?
A. solanicola mitochondrial genome
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Thank you!PBCRC Bacterial Pathovars (PBCRC2156)
Rebekah Frampton
Sarah Thompson
Falk Kalamorz
The Plant Micro Group
Debbie Kent
Isabel Valenzuela-Gonzalez
Gary Taylor
Piotr Trebicki
Jessica Dohmen-Vereijssen
Project team (PBCRC 62116)
Brendan Rodoni
Rachel Mann
Jason Shiller
Grant Smith
Alan Yen