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The nature of resistance to
Neonectria ditissima in
apple species
Antonio Gomez-Cortecero and Amanda Karlstrom
There is no clear evidence of how N. ditissima penetrates into the host
Necrotrophic lifestyle with a sophisticated mechanism of infection
• Cell wall degrading enzymes
• Proteins secreted from the pathogen that alter host processes
The mechanism by which the pathogen is detected by the host it is still
unknown
C141 – PhD on the molecular basis of pathogenicity of N. ditissima.
C141 – PhD on the molecular basis of pathogenicity of N. ditissima.
Extend and deploy an accurate and quantifiable pathology test to measure
quantitative differences in pathogenicity of differences N. ditissima isolates
Develop genomic resources and conduct a gene expression analysis in order
to identify and characterise pathogenicity genes involved at different stages
of the infection
Once candidates have been identified, validation of gene function by targeted
disruption of the pathogen
Aims
Pathogenicity tests
Leaf scar inoculation
• One year old grafted trees
• Five leaves removed randomly along the tree
Cut shoot test
• One year old dormant shoots
• Day/night: 22 ˚C/16 ˚C and 20 hour/4 hour
• 80% humidity
• Three axillary buds were inoculated
• Buds cut off with a scalpel to simulate lead
petiole scar
Robusta 5 Gala Golden Delicious
Results
Different components of resistance?
Abiotic stresses in modulating plant resistance?
Area Under Disease Progress Curve. The AUDPC measures the disease throughout a period.
Leaf scar test Cut shoot test
More resistant
More susceptible
Apple seedling inoculation
Seven month old apple seedlings
were inoculated with N. ditissima
Eleven different crosses were tested
(16 plants per cross)
N. ditissima isolate R09/05 (2.7x105
spores/ml)
Temperature/Humidity: 20°C/80%
Apple seedling inoculation
Crosses MDX053 (Aroma x Fuji) and
MDX051 (Gala x Santana) had the lowest
median AUDPC values/most resistant
Crosses MDX057 (Gloster 69 x Idared) and
MDX068 (Grenadier x Golden Delicious) had
the highest median AUDPC values/most
susceptible
MDX068 Grenadier (I) x Golden Delicious (R)
MDX054 Aroma (I) x Golden Delicious (R)
MDX052 Aroma (I) x Gala (S)
The nature of resistance differ between
cultivars?
Gene expression patterns (work in progress)
A list of pathogenicity genes has been generating analysing the genome of N. ditissima
Certain areas of the genome contain
multiple genes involved in the pathogenicity
of N. ditissima
Analysis of gene expression during
infection will help narrow down those
regions
Validation of gene function will be
attempted by knockout of key genes of the
pathogen
Whether differences in an isolate’s
virulence are correlated with sequence
variation will be further investigated
Resistance - what we know
Approx. 50% of apples produced in the UK are
cultivars derived from two progenitor apple varieties –
Cox and Gala – both susceptible to N. ditissima
Many common rootstocks are susceptible, with M9 still
the predominant rootstock being highly susceptible
Natural resistance is poorly deployed in modern
rootstock and scion breeding programmes
Complex nature of resistance – making selection
without molecular markers difficult
rhs.org.uk
New BBSRC-LINK grant
New BBSRC-LINK grant for work on the nature of
resistance to fungal canker in apple species.
Started January 2017
The overall project objective is to elucidate the
genetic basis of resistance to canker in order to
develop molecular markers to be used in
developing new varieties
Work will focus on resistance in both rootstocks
and scions
New BBSRC-LINK grant
Identification of resistance in five scion crosses and one
rootstock cross
Assess several crosses using cut shoot, seedling, fruit
and leaf scar infection techniques to establish the link
between controlled tests, tissue-specific resistance and
effective field resistance
Identify genes and genomic regions involved in
resistance towards fungal canker
Transformation of candidate resistance genes into a
susceptible variety to validate gene function
Work packages
WP1: Genome sequencing,
genotyping and resistance gene
enrichment of parental clones
WP2: Identification of quantitative
resistance in the cultivated apple
germplasm
Leaf scar test on small grafted plants
Cut shoot test
Six field sites (3 grower and 2 East Malling)
Artificially inoculated fruit tests
Genome sequencing of parents
Genotyping of multiple scion
crosses and one rootstock cross
Identification of potential resistance
genes in parents
Work packages
WP4: Validation of resistance
responsesWP3: Identification of resistance in
other Malus species for rootstock
improvement
Leaf scar test on small grafted plants
Cut shoot test
Field assessment of canker expression
Transformation of candidate resistance genes into
susceptible variety
Validation of resistance in the transformed variety
Summary
Mechanism of effective field resistance is not fully queried in
pathology tests
Potentially complex nature of inheritance of resistance to fungal
canker
New pathology tests and field assessments to confirm the
consistency in the initial results
Assessment of resistance in rootstock material
Identify gene regions involved in resistance to canker
Validation of the effect of resistance genes through transformation
into a susceptible variety