Psyllid microflora: Implications for liberibacter disease surveillance and pest control

biosecurity built on science

Jacqueline Morris

PhD Candidate

Plant Biosecurity Cooperative Research Centre

Psyllid microflora- Implications for liberibacter disease surveillance

and pest control


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


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)


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.


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)


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


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


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


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


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


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


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


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


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


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


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

Date post:	23-Jan-2018
Category:	Science
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Psyllid microflora: Implications for liberibacter disease surveillance and pest control

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