The nature of resistance to Neonectria ditissima in apple species

Lead Research Organisation: National Inst of Agricultural Botany
Department Name: Centre for Research


In the UK, land devoted to modern apple production is rising at a rate of between 7 and 10% per annum, however (similar to the global situation) the combination of more intensive planting of canker-susceptible cultivars has led to an increase in losses due to the fungal pathogen Neonectria ditissima. Problems in both in-field production (tree death and yield reduction) and post-harvest losses (due to postharvest rot), call for an innovative approach to combat this disease.

Approximately 50% of apples produced in the UK are cultivars derived from two progenitor apple varieties, Cox and Gala, both of which are susceptible to fungal canker, indeed these newer varieties such as Kanzi, Rubens and Jazz are extremely susceptible to canker, which has led to large problems in orchard establishment and increasingly post-harvest losses, especially in wet years. In severe cases, in field yield decline can reduce potential pre-harvest yield by at least 25% due to loss of fruiting wood and 5% of postharvest apple crops are lost due to Neonectria infection. There is poor chemical control of canker of trees due to its systemic nature of the disease. It has been shown that the pathogen is often present in the plants from a young age (in the nursery) and that both the rootstock and the scion can be infected, but remain asymptomatic for long periods of time.

Natural resistance to canker has been well documented, but extremely poorly deployed in modern rootstock or scion breeding programmes. The reasons for this can be attributed to the complex nature of resistance (making selection without molecular markers difficult), the cost of mass screening to eliminate highly susceptible individuals, different types of resistance (all of which are uncharacterised), the relatively recent re-emergence of the problem and the potentially heterogeneous nature of the pathogen.

This proposal will use cutting-edge genomics approaches to identify natural plant-derived resistance to this important pathogen in both scion varieties and in rootstocks. In this project, a multi-parental population will be used to characterise resistance and a combination of targeted genome sequencing techniques and pedigree-based genotyping will be used to accurately narrow down the specific genes responsible for resistance. Once obtained, in proof of principle experiments, genes underpinning the resistance will be transferred into a susceptible cultivar, in order to validate the resistance in other genetic backgrounds.

The industry are supporting this proposal through the LINK scheme, as they recognise the need for a comprehensive strategy to identify the genetic basis of disease resistance, for use in marker assisted breeding (MAB) and other breeding techniques.

Technical Summary

Understanding the genetic basis of resistance to fungal canker is important due to the need for resistant cultivars for global production. In order to fulful this requirement a programme of research querying quantitative resistance in the cultivated apple and species-level resistance in apple roostocks is proposed. Apple (Malus x domestica) is an outcrossing species with a highly heterozygous genome. Using the latest, PCR-free library preparation techniques, along with cutting-edge genome assemblers (DISCOVAR) founding parents of a multi-parental set of lines and mapping population parents will be sequenced. Although QTL mapping will be unbiased, in order to accurately capture the highly variable resistance gene space, (for this and for other disease resistance studies), both NB-LRR and eLRR families will be queried using a PacBio-RenSeq approach to resolve phased candidate resistance genes, without recourse to a BAC library. Genotyping the multiparental population using 20k illumina SNP chips (and the rootstock population with GbS) will allow full imputation of genome sequences onto individual clones. Phenotyping replicated progenies, using a variety of pathogenicity tests, from cut shoot tests, fruit tests and whole tree infection in multiple environments will allow QTL mapping to be carried out to identify the genetic basis of resistance. Further analysis of RNAseq data of infection will allow the role of different candidate genes underlying high resolution QTL to be studied. Validation of single and multiple candidate genes using multi-gene GoldenGate constructs transformed into susceptible lines will allow validation of QTL; promoter analysis will elucidate tissue specificity of resistance responses. This will also facilitate further characterisation of candidate pathogenicity genes in Neonectria, from ongoing collaborative pathogen genome sequencing work.

Planned Impact

This grant will have a global impact, both on the research field internationally and on the international industry, especially the UK industry. Through full engagement with industry stakeholders, maximum translation of this research will be ensured, driving forward the UK plant breeding industry in a globally competitive market.

Direct beneficiaries:

1. Commercial private sector
The UK and international plant breeding sector will benefit enormously from this endeavour and will allow these industries to first develop better markers for QTL (most traits in apple are quantitative and multiple QTL underpin these) and move from marker level associations to candidate gene associations. This is important for next-generation genome editing approaches and functional validation of candidate genes. This moves the industry very quickly to a point where pedigree-based selection and genome-wide selection are affordable and tractable options for crop improvement. Placing this in the hands of the UK partners will give the UK business a significant competitive edge.

2. Fruit growing sector in the UK
UK industry will benefit as they will be able to access a resource that is beyond their means to create. Longer term it is anticipated that the UK partners will make significant use of this resource and knowledge generated from this pre-competitive work. This may lead to further competitive work funded by other research bodies (e.g. innovate UK or AHDB). Advancing genomic resources in horticultural crops is a key aim of the AHDB-Horticulture and evidenced by their support in this proposal (Benefit within 5-10 years).

3. Public and retail sector
Several UK retailers aim to double sales of UK-produced fruit by 2020; this project will assist that aim and improve UK productivity and competitiveness. Downstream science conducted utilising the resources generated in this project will lead to more reliable production methods and potentially reduce wastage in the supply chain (through reduced inputs and better variety development) (Benefit within 5-10 years).

Indirect beneficiaries
The wider apple growing industry (UK and beyond)
As a result of resistance markers to fungal canker, the rate of change of varietal development will increase, leading to greater benefits to downstream growers, packers and producers.

Government, public and policy benefits
The public will benefit, not only from the improved position of UK agribusiness (and access of breeders to novel technologies), but also through the long term improvement in supply chain resilience through improved cultivar development. In the longer term the public will benefit through increased food security and sustainability, as a result of scientific improvements on horticultural crops. This feeds in to many UK Government and EU policy agendas including: health (improving produce quality, pesticides (reducing residues through improved resistance), water (ability to grow nearer water courses), climate (growing crops perennially will improve carbon sequestration) and environment (reduced carbon and pesticides) (Benefit within 5-10 years).
Description This work is still underway, but we have discovered multiple sources of resistance to N. ditissima that are being characterised genetically.

2019- update

This project is having great success at identifying resistance to fungal canker disease in apple. Predictions appear to be very strong and the genetic basis is highly heritable indicating a clear way forward.

An initial publication is being prepared.

2020 update- This work is progressing very well and publications are nearing completion. Strong resistance has been found and this work and will undoubtably be used. Some covid delays have been experienced in planting field trials in Northern Ireland. However, we have tried to overcome this issue.

2021- update- A preprint of our first reports of quantitative resistance has been submitted in biorxiv. This work has been very successful in uncovering the quantitative resistance in apple to Neonectria. We have now carried out RNAseq of resistant and susceptible lines and found significant differences in activation of resistant responses, which we are in the process of trying to link to observed quantitative genetic variation. We are close to completing a wider multi-site analysis of resistance, which will further validate our previous resistance discoveries and also lead to an understanding of durability/stability of resistance. We have carried out further RNAseq in order to try to narrow down candidate genes, which with our original strategy has been challenging. We have validated that CRISPR Cas9 is working in apple and have this capability for further work, though we have not been able to progress to candidate knockouts as originally hoped, as despite clear differences in resistant/susceptible lines at the transcriptome level, we are struggling to link to any single candidate gene. We hope that our expanded transcriptome analysis will enable mechanistic characterisation in the future.
Exploitation Route Our findings are already being integrated into breeding pipelines.
Sectors Agriculture, Food and Drink

Description Again, work is underway, but our findings are being used in breeding programmes already. 2019- update. The level of integration into breeding programmes is increasing and inward investment is being sought to scale up activity to enhance the global reach of this work. 2020-21 update As with previous years movement of knowledge from research to delivery has continued. The commercial breeding programme now has high quality genomic predictions of quantitative resistance available to it and information on predicted resistance has been delivered to industry partners.
First Year Of Impact 2020
Sector Agriculture, Food and Drink
Impact Types Economic

Title Protocol for phenotyping of disease resistance to Neonectria ditissima in apple 
Description Developed methodology to obtain reproducible estimates of host resistance to the fungal pathogen Neonectria ditissima in apple trees 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2022 
Provided To Others? Yes  
Impact Phenotyping method has been used within our research institute to determine the resistance of new apple varieties on behalf of a fruit cooperative This has been done to inform whether the new variety will be widely adopted and planted by the cooperative. 
Description Collaboration with AFBI 
Organisation Agri-Food and Biosciences Institute
Country United Kingdom 
Sector Public 
PI Contribution We have set up a collaboration to extend our phenotyping out to NI as this has value for all partners.
Collaborator Contribution They are hosting a trial of our genomic selection population for disease phenotyping
Impact This will result in improved genomic predictions
Start Year 2019
Description Collaboration with Eric van de Weg, Wageningen University and Research 
Organisation Wageningen University & Research
Country Netherlands 
Sector Academic/University 
PI Contribution NIAB has contributed to this collaboration through the provision of fungal isolates and phenotyping protocols
Collaborator Contribution Wageningen Unviersity and Research have contributed with expertise in data analysis in the software FlexQTL and with preparing a manuscript for publication
Start Year 2017
Description Collaboration with New Zealand PfR 
Organisation Plant & Food Research
Country New Zealand 
Sector Private 
PI Contribution Joint sampling programme for global isolates of Neonectria
Collaborator Contribution Contribution of genome sequences
Impact Ongoing research into genome evolution of Neonectria Funding for travel (2020 BBSRC grant)
Start Year 2017
Title Molecular markers/ genomic prediction for disease resistance 
Description Genomic prediction/ molecular markers for canker resistance identified. This was the primary outcome of the grant that the industry partner required. 
IP Reference  
Protection Protection not required
Year Protection Granted
Licensed Commercial In Confidence
Impact This critical knowhow is now embedded in an industry funded breeding programme which is developing a pipeline of disease resistant varieties to the market.
Description AHDB Tree Fruit Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Oral presentation on technical information day for fruit growers, agronomists and other people in the horticultural industry.
Interest shown in how breeding can be used to reduce incidence of European canker in commercial apple production.
Year(s) Of Engagement Activity 2017
Description Apple canker workshop at Yangling A&F University 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Attendance and contribution at a workshop associated with BBSRC China Partnering funding on apple canker research. This allowed presentation on Apple canker research at NIAB EMR, involvement in discussions on European and Asian apple cankers and also highlighting genomic research performed across other projects at NIAB EMR associated with development of molecular diagnostics. Genomic work performed on apple canker at NIAB EMR led to discussions on joint publications between groups. Discussion on Fusarium and how we have developed molecular diagnostics from that work led to discussion on further research projects.
Year(s) Of Engagement Activity 2018
Description Article in AHDB news on apple and cherry canker 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Wrote an article highlighting the problems and the funded solutions. This raised awareness of the funded work and offered growers the option to get in touch and add their farm to a study.
Year(s) Of Engagement Activity 2017
Description BSPP Plant Pathogen Diagnostic Course 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Hosted a one day practical course on Plant Pathogen Diagnostics - From symptoms to sequence. We covered a range of pathology skills and knowledge. Collated feedback of the day rated the course very useful, with over 90% of delegates stating they were satisfied or very satisfied with the content, speakers and organisation of the course.
Year(s) Of Engagement Activity 2018
Description Fourth International Horticulture Research Conference 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Hosted the Fourth International Horticulture Research Conference at NIAB EMR. The conference brought together international researchers conducting basic research on horticultural crops, facilitating them to showcase their latest research findings and to network with colleagues from around the world. The conference allowed us to raise the profile of NIAB EMR and the research we are conducting. A poster was presented specifically detailing the work in this project.
Year(s) Of Engagement Activity 2017
Description Fruit Focus 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Fruit Focus is the leading technical event for the soft fruit industry, with ~1,200 attendees. Several posters were produced for the event and were presented throughout the day, which sparked questions and discussion. Attendees reported increased knowledge of strawberry pathogens and the work we are doing to improve resistance to them.
Year(s) Of Engagement Activity 2018
Description Hosted representatives from Worldwide Fruit Ltd and M&S 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Staff from the fruit marketing and distribution company Worldwide Fruit Ltd and supermarket M&S visited the research organisation both for formal presentations about apple canker and visits to the orchards.
Year(s) Of Engagement Activity 2021
Description Presentation at industry workshop in Belgium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact 20-30 representatives working within the apple industry across Europe attended a presentation on the role of plant host resistance to reduce European canker disease during a workshop. Resulted in discussion on the importance of testing the susceptibility to this disease in new varieties before they are widely planted and has led to further collaboration with an industry partner and colleagues at Wageningen University.
Year(s) Of Engagement Activity 2019