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

Acknowledgments

Maria Carcamo

Marzena Lipska

Dr. Maria Sobczyk

Dr. Helen Bates

Dr. Charlotte Nellist

Felicidad Fernandez

Dr. Xiangming Xu

Dr. Richard Harrison

Dr. Robert Jackson

Dr. Andrew Armitage

Dr. Robert SavilleThank you for your attention