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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
biosecurity built on science
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
biosecurity built on science
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)
biosecurity built on science
Zebra Chip
biosecurity built on science
Zebra Chip
biosecurity built on science
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
biosecurity built on science
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
biosecurity built on science
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
biosecurity built on science
Discovery of the putative pathogen in New Zealand
January 2008, a new disease observed in glasshouse tomato and capsicum crops in New Zealand
biosecurity built on science
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
biosecurity built on science
Investigation microscopy
TEM of thin sections undertaken by Plant & Food Research
Phloem-limited bacterium-like organisms (BLOs) observed
biosecurity built on science
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
biosecurity built on science
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
biosecurity built on science
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)
biosecurity built on science
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’.
biosecurity built on science
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
biosecurity built on science
Zebra Chip in New Zealand
biosecurity built on science
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
biosecurity built on science
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
biosecurity built on science
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
biosecurity built on science
Diagnostics
biosecurity built on science
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
- +
biosecurity built on science
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
biosecurity built on science
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
16S rRNA diagnostics of Ca. L. solanacearum
biosecurity built on science
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
16S rRNA diagnostics of Ca. L. solanacearum
biosecurity built on science
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
biosecurity built on science
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
biosecurity built on science
In-field diagnostics
biosecurity built on science
In-field diagnostics
biosecurity built on science
In-field diagnostics
biosecurity built on science
Diversity
biosecurity built on science
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
biosecurity built on science
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
biosecurity built on science
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
biosecurity built on science
Host Plants
biosecurity built on science
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)
biosecurity built on science
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
biosecurity built on science
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
biosecurity built on science
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
biosecurity built on science
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
biosecurity built on science
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
biosecurity built on science
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 built on science
Biosecurity Lessons
biosecurity built on science
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)
biosecurity built on science
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
biosecurity built on science
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)
biosecurity built on science
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