Zebra Chip disease of potato - AUSVEGCandidatus Liberibacter solanacearum (CLso) Putative casual agent of Zebra Chip of potato Synonym Ca. L. psyllaurous (Hansen et al. 2008) α-Proteobacterium

biosecurity built on science

Zebra Chip disease of potato

Rebekah Frampton

The New Zealand Institute for Plant and Food Research Limited, Lincoln, New Zealand

Hort Connections, May 2017, Adelaide Convention Centre, Adelaide, Australia


Thanks and acknowledgments

Grant SmithJessica Dohmen-VereijssenIan ScottSarah ThompsonKerry SullivanFalk KalamorzAnna-Marie BarnesShirley ThompsonRuth ButlerNatasha AgnewDavid LoganAleise PuketapuMano Sandanayaka

Kyla FinlayKevin PowellIsabel ValenzuelaAlan Yen

Chris JohnsonAimin WenNeil Gudmestad

Lia Liefting and colleagues

Collaborators in the larger international CRC2002/ CRC2156 projects developing and deploying genomics-based validated diagnostic protocols for a range of bacterial genera


Thanks and acknowledgments

Funding• Plant & Food Research (NZ)• Plant Biosecurity Cooperative Research Centre (AUS, NZ, USA)• USDA-NIFA-SCRI (USA)• MPI (NZ)• MBIE (NZ)• Sustainable Farming Fund (NZ)


Zebra Chip


Zebra Chip


Zebra Chip history

1994: Symptoms first reported from near Saltillo, Mexico• Symptoms include stunting, swollen nodes, chlorosis, aerial

tubers, leaf scorching, medullary ray streaking

Early 2000s: First noted in Lower Rio Grande Valley/ Pearsall, Texas

By 2006: Disease had spread to most potato production areas of Texas, Kansas, Arizona, Colorado, Nevada, New Mexico, and California

2008: Reported in New Zealand

2012: Reported from Idaho, Oregon and Washington State

From SCRI 2014 Calendar From SCRI 2014 Calendar


Association of psyllids with Zebra Chip in the USA

Joe Munyaneza (USDA-ARS) and colleagues

Symptoms of Zebra Chip suggested a phytoplasma aetiology

Phytoplasma vectors were collected from the field

• Sternorrhyncha (suborder of the Hemiptera) contains psyllids, aphids, whiteflies

and mealybugs

• Aphids, whiteflies and mealybugs are efficient vectors of plant viruses and

bacteria such as phytoplasmas

• Several examples of psyllids vectoring phytoplasmas (eg pear decline, apple

proliferation)

• Nault (1997): no confirmed reports of transmission of any plant virus by any of

the 2,000 or so species of psyllid


Association of psyllids with Zebra Chip in the USA

Joe Munyaneza (USDA-ARS) and colleagues

Symptoms of Zebra Chip suggested a phytoplasma aetiology

Phytoplasma vectors were collected from the field

High populations of Tomato Potato Psyllid (TPP; Bactericera cockerelli) noted

Experiments established link between psyllids and Zebra Chip

• Psyllid yellows

• At this time no association of a pathogen with the disease


Discovery of the putative pathogen in New Zealand

January 2008, a new disease observed in glasshouse tomato and capsicum crops in New Zealand


Investigation of the aetiology

Plants were tested for a range of pathogens• Pathogenic fungi and culturable bacteria

• Generic tests for viruses

o Herbaceous indexing

o Transmission electron microscopy (leaf dip)

o dsRNA purification

• PCR tests for phytoplasmas, viruses, viroids

TPP (B. cockerelli) observed in association with affected crops

All negative


Investigation microscopy

TEM of thin sections undertaken by Plant & Food Research

Phloem-limited bacterium-like organisms (BLOs) observed


Identification of the BLO in New Zealand

Universal 16S rRNA primers (fD2/rP1) used in combination with a range of specific 16S rRNA PCR primers

Healthy Symptomatic No templatecontrol • A unique 1-kb fragment amplified from

symptomatic plants only

• 97% identical to 16S rRNA gene of Candidatus Liberibacter asiaticus

• Phylogenetic analyses of entire 16S rRNAgene and partial rplKAJL-rpoBC operon determined it was a new Liberibacterspecies – Candidatus Liberibacter solanacearum


Candidatus Liberibacter solanacearum (CLso)

Putative casual agent of Zebra Chip of potato

Synonym Ca. L. psyllaurous (Hansen et al. 2008)

α-Proteobacterium

Unculturable

Psyllid (Hemiptera: Triozidae) associated and vectored

Five molecular/biological haplotypes described to date

Currently one of eight named species in the genus

• Seven unculturable

• One culturable


Ca. L. solanacearum

Five CLso haplotypes (A to E)• Based on SNPs and indels in 16S rRNA, 16S/23S ISR (intergenic spacer region) and 5S rRNA regions• Broad plant host family differentiation (AB/ CDE)• Broad geographic differentiation (AB/ CDE)• Limited vector differentiation (AB/ C/ DE)

Highly likely more variants to emerge/ be discovered

Haplotype Region Vectors (Hemiptera: Sternorrhyncha) Plant Host Family

A Americas, New Zealand B. cockerelli (Psylloidea: Triozidae) Solanaceae (various species)

B Americas B. cockerelli (Psylloidea: Triozidae) Solanaceae (various species)

C Scandinavia Trioza apicalis (Psylloidea: Triozidae) Apiaceae (Carrot)

D Europe/ Africa Bactericera trigonica (Psylloidea: Triozidae) Apiaceae (Carrot)

E Europe/ Africa Unknown (possibly B. trigonica) Apiaceae (Celery and Carrot)


The (Candidatus) Liberibacter genus

Liberobacter proposed by Jagoueix et al. in 1994

• Unculturable phloem limited BLO associated with citrus greening

Liberibacter first used by Garnier et al. in 2000

• Described a BLO in found in Calodendrum capense (Rutaceae)

• Liberobacter was determined to be orthographically incorrect

Candidatus

• Murray & Schleifer (1994) proposed the category Candidatus ‘to provide a proper record of sequence based potential new taxa at the genus level’ to resolve the problem that ‘formal names are being proposed for uncultivated prokaryotes whose uniqueness is defined only by very limited characteristics’.


Currently described (Candidatus) Liberibacter speciesYear first described Psyllid Vectors Plant Host Family

Ca. L. asiaticus 1994 Diaphorina citri, Trioza erytreae, Cacopsylla citrisuga

Rutaceae (various species)

Ca. L. africanus 1994 D. citri, T. erytreae

Ca. L. americanus 2004 D. citri

Ca. L. caribbeanus 2015 D. citri

Ca. L. solanacearum(syn Ca. L. psyllaurous)

2008 Bactericera cockerelli, Trioza apicalis, Bactericera trigonica

Solanaceae (various species),Apiaceae (Carrot, Celery)

Ca. L. europaeus 2011 Arytainilla spartiophila, Cacopsyllapyri

Scotch broom, Pear

L. crescens 2012 None described None described

Ca. L. brunswickensis 2017 Acizzia solanicola None described


Zebra Chip in New Zealand


2006: B. cockerelli found in New Zealand

2008: Zebra Chip in New Zealand

2008: Ca. L. solanacearum found in New Zealand

2008-2009: Cost to potato industry NZ$47-56 M • Increased insecticide application

2009-2010: Average $700/ha extra agrichemicals

2010-2011: Cost to potato industry NZ$28 M• Includes NZ$6 M for pest control

Management requirements varied between North and South Islands

Zebra Chip in New Zealand

Ogden 2012 SCRI Zebra Chip Reporting Session


Many tamarillo growers have left• 80 in five years

Field tomatoes • High numbers of B. cockerelli in Hawke’s Bay

• Spray programmes generally effective

Glasshouse tomatoes• Aggressive removal of infected plants

• 4-6% yield loss

• 2011: NZ$5 M in control costs

Ca. L. solanacearum and B. cockerelli in New Zealand

Ogden 2012 SCRI Zebra Chip Reporting Session


Different Zebra Chip symptoms and severity in New Zealand compared to the US• Less dominant striping in fried slices

• Different biological results

• Infected tubers sprouted (sensitivity of assays)

• Different described epidemiology

• Cultivar, environment, cultivation practices, vector behaviour

• The more ‘aggressive’ variant is not present in NZ

• Limited genetic diversity of CLso in NZ?

New Zealand (Pukekohe)USA (Texas)

Zebra Chip symptoms


Diagnostics


16S rRNA common diagnostic and classification target

Liberibacter genomes contain three copies of the rRNAoperon

Initial PCR assays targeted the 16S rRNA region (Liefting et al.)• Titre not quantifiable (end point)

• Sampling issues

• Sensitivity

16S rRNA diagnostics of Ca. L. solanacearum

Size marker

- +


Different samples from the same infected plant did not consistently show the presence of Ca. L. solanacearum using PCR

Low titre of bacteria in potato tissue?

Uneven distribution of bacteria in potato tissue?

P2AP1BP1A P2B P3A P3B + + -

Diagnostic sampling effects


Liberibacter genomes contain three copies of the rRNAoperon

Initial PCR assays targeted the 16S rRNA region (Liefting et al.)• Titre not quantifiable (end point)

• Sampling issues (bacterial distribution in plant)

Semi-nested PCR assay developed (16S rRNA) (Beard et al.)• Qualitative (end-point) or quantitative (qPCR)

• Normalised (genomic units/ microgram gDNA)

• Up to 50x more sensitive than end-point PCR



Cross-reactions with plant chloroplast and other bacteria

Difficult to specifically identify pathogen• Ca. L. solanacearum haplotype A

• Pseudomonas syringae pv. actinidiae biovar 3

Can we use other regions of the genome?

Healthy Symptomatic No templatecontrol



CLso-NZ11.31 Mbp

Red = selected loci Green = prophagePurple = ‘phage remnant’ regions

PBCRC 2002 + 2156 Sequenced genomes Identified unique regions

Many targets are in/ near to ‘phage remnant’ regions

Hypothetical proteins

Designed primers Tested, tested, tested

Genome based diagnostics


Better surveillance and better response tools, technologies, systems, strategies to improve the probability of a successful eradication

Laboratory based diagnostics

On-orchard/ In-field diagnostics

Earlier identification earlier response • Surveillance

• Eradication

• Monitoring

• Management

Diagnostics for Ca. L. solanacearum


In-field diagnostics






Diversity


Teulon DAJ, Workman PJ, Thomas KL, Nielsen MC, Zydenbos SM. 2009. Bactericera cockerelli: incursion, dispersal and current distribution on vegetable crops in New Zealand, p. 136–144.

2006: B. cockerelli found in New Zealand

2008: B. cockerelli in all major potato growing regions

2008: Ca. L. solanacearum found in New Zealand

Management requirements and Zebra Chip symptoms varied between North and South Islands

Ca. L. solanacearum and B. cockerelli in New Zealand


Selected 3 genome regions that were variable in initial testing of USA and NZ samples

All 29 New Zealand CLso samples contained all 3 loci

Suggests limited genetic variability in CLso over time, location or host in New Zealand• There were no Californian CLso samples in the initial American screen

• New Zealand assessment limited by availability of DNA extracts

Origin of the CLso in New Zealand remains uncertain• One or more inclusions from the same geographic location more likely than separate incursions

from different regions

• Still remains unresolved whether genomic differences contribute to the differences in Zebra Chip pathology between New Zealand and the USA

Ca. L. solanacearum diversity


Network analysis of mitochondrial genomes

Each circle is a different sequence type

Circles links based on DNA sequence changes

Size of the circle represents number of samples

Geographical split Overlap between New Zealand

and California Lacking samples from USA,

especially California

B. cockerelli diversity


Host Plants


What other plant hosts for B. cockerelli are out there?

TPP host plant in Solanaceae• these are widespread in Australia and New Zealand: crops and weeds, cultural uses too

Knowledge gap: ecology of TPP and CLso related to their non-crop host plants (temporal & spatial dynamics, feeding, development)

http://www.flickr.com/photos/16921893@N00/8443518962


Host plant surveys around crops

Host plants of TPP and CLso are not restricted to crop species, and include weed species, which provides challenges for surveillance, eradication and management• All TPP life stages were present on non-crop host plants throughout the year

• So they are not alternative hosts, but hosts

• Jerusalem cherry and thorn-apple tested positive for CLso in Hawke’s Bay


Spatiotemporal dynamics of TPP throughout the year

There was a low background population of B. cockerelli flying around in the environment

When African boxthorn was present adjacent to a crop, there was increased activity nearby and an edge effect may be observed in the host crop

B. cockerelli multiplied in the crop but did not disperse far

A desiccated crop increased adult flight in B. cockerelli


Key outputs for this project

Knowledge • Scientifically validated list of crop and non-crop alternative hosts in Australia and New Zealand

Tools & training• Targeted monitoring and weed management advice to growers, plant primary industries and

biosecurity agencies


Feeding of B. cockerelli on tomato and boxthorn

Host plant species alone was not decisive in determining the number and duration of phloem salivation (E1) and ingestion (E2) events in B. cockerelli

CLso infection status of B. cockerelli was more important in determining feeding behaviour• CLso-positive B. cockerelli are more likely to salivate than CLso-negative ones

• CLso-negative B. cockerelli are more likely to ingest phloem sap than CLso-positive ones


TPP performed best

TPP performedworst

Days till first egg laid

Potato Boxthorn Poroporo Tomato

Number of eggs/female/day of life

Poroporo Boxthorn Potato Tomato

Mortality overall Poroporo Potato Boxthorn Tomato

Days to female death

Poroporo Potato Tomato Boxthorn

Days to male death

Poroporo Potato Boxthorn Tomato

Development time overall

Poroporo Potato Boxthorn Tomato

Development and fecundity of B. cockerelli on host plants


This has implications for• biosecurity preparedness plans and pest risk assessments

• surveillance and monitoring (techniques and locations)

• incursion responses

• pest and disease management

Non-crop host plants are important in the ecology of B. cockerelli


Biosecurity Lessons


Ca. L. solanacearum: some biosecurity lessons

Responding to an incursion of unknown aetiology is hard work• Immediate trade implications, substantial immediate costs

• Linking a molecular signature to the causal agent is important

• Allows delimiting surveys, and initial identification of candidates for the causal agent

Understanding the epidemiology of a vectored biosecurity pathogen is critical to an effective, sustainable response• Insect and plant hosts (vertical and horizontal transmission)

• Targeting the vector is not necessary targeting the pathogen

• Focus on the insect and the pathogen (transmission type)


Ca. L. solanacearum: a few more biosecurity lessons

Everyone wants a solution: now!• Pressure for immediate answers and solutions

• The big picture is blurred: hard to ascertain the value and context of new results when the biology is not clear

o Transmission characteristics

o Conflicting results from assays with different sensitivity thresholds

Good applied research needs a solid base• Sustainable outcomes are delivered from understanding

• Essential strategic basic research needs to be done whilst responding


Who will benefit from this research?

Growers (potato, tamarillo, capsicum, chili, eggplant, tomato)

Plant primary industries NZ & AU

Researchers US, NZ, AU • CRC2002/2156, CRC2146, NZ Govt funded TPP/CLso programme

Biosecurity decision makers / Surveillance / Pest Risk Assessment / Diagnostics• PHA, SPHD, SNPHS

• Internationally (especially for PRAs)


Thank you

Further information:Rebekah Frampton [email protected]

Grant Smith [email protected]

Jessica Dohmen-Vereijssen [email protected]

Dr. Jessica Lye

AUSVEG Manager – Science and Extension

AUSVEG Vegetable and Potato Biosecurity Officer

03 9882 0277

0401 555 567

[email protected]

mailto:[email protected]



Documents

Zebra Chip disease of potato - AUSVEGCandidatus Liberibacter solanacearum (CLso) Putative casual agent of Zebra Chip of potato Synonym Ca. L. psyllaurous (Hansen et al. 2008) α-Proteobacterium