European canker: a devastating disease for young apple orchards

in Northern Europe

Larisa GustavssonPlant Breeding and Biotechnology

Alnarp

European canker

• A serious disease which affects more than 100 species• Caused by a fungal pathogen, Neonectria ditissima (formerly

Nectria galligena), which is able to cross-infect a wide range ofhosts

M. Lateur

• The fungus uses both (ascospores and conidia) to enter the host• Damages side branches and the main trank• The infection is spred aerially and with rain splash

Perithecia contain ascospores (genetic recombination)

Sporodochia contain conidia (vegetative reproduction of a fungus)

Marc Lateur

Agrios, 1997

Disease cycle of canker caused by Neonectria ditissima

Sporodochium

Part of a conidiophore from a sporodochium produced on apple bark

Macroconidia of N. ditissima

The pathogen is a wound parasite• Exploits wounds caused by external damage• Able to exploit natural ruptures which occur during the normal

growth of the plant:- leaf scars- fruit scars- cracks- frost injury- pruning wounds- wounds caused by insects- wounds caused by other pathogens

Can invaide the xylemfibers and stay dormant M. Lateur

What happens in the plant?

• In the presence of the pathogen the host forms a protective phellogen layer, which stops the pathogen for some time, but after a while this protection is broken down

• The host forms a phellogen barrier some distant away from the • The host forms a phellogen barrier some distant away from the pathogen in attempt to enclose it again

• Once the pathogen has entered the stem, its growth progresses rapidly and it can in short time encircle the entire stem

• Initial cankers – sunken areas in the bark• Edges of the canker swell due to increased phellogen activity• Lesions extend more rapidly in a longitudinal direction than in

transverse• Pathogen spreads through the tissue – kills the host’s cells • Annual formation of wound phellogen and callus -> typical circular

target pattern

Neonectria ditissima fungus damages also fruit during storage

http://www.inra.fr/hyp3/images/6034013.jpg

Synonym: Cylindrocarpon mali

Marc LateurWalloon Agricultural Research Centre CRA-W, GEMBLOUX - BelgiumDept. of Life SciencesBreeding & Biodiversity Unit

Plaies de liens = 0,8%Wounds after branch

Wounds after tree ties

Plaies de taille = 13,1%

Aisselles de branches =

6,6%

Cicatrices fruitières =

10,1%

Cicatrices foliaires =

69,4%

= 0,8%

Leaf scars

Fruit scars

Angle of branches

Wounds after branch prunings

HOW DOES IT LOOK IN SWEDEN????

’Evaluation of useful diversity in Swedish apple: resistance to European canker’SJV, 2012

• Cankers, formed on minor branches and side shoots can be removed by pruning -> time consuming, high labour costs

• Often not sufficiently successfull, since the disease is of a systemic character

• Fungicides can reduce the spread of cankers, but none of the fungicides is able to eradicate the fungus or cure trees with established infections

European canker is regarded as the potentially most European canker is regarded as the potentially most devastating disease for Swedish and North European apple production

M. Lateur

The disease is especially dangerous for young apple trees…

• Can be infected already in nursery• Usually the main trank is damaged and the whole tree dies. The

disease can devastate the whole plantation of newly planted trees• Growing of scab-resistant cultivars can increase problem with • Growing of scab-resistant cultivars can increase problem with

European canker, since fungicides applied against scab are also effective to Neonectria

M. Lateur

THEREFORE…

• In Sweden and Northern Europe there is a need to breed new apple cultivars with high level of resistance to European canker

• It is important to find especially resistant cultivars for use as parents within the apple breeding programs but also for direct use in apple growing. Genetic dissection of this trait is important for development of molecular markers to be used in breeding, understanding molecular mechanisms underlaying resistance as well as identification of candidate genes

• There are considerable differences among cultivars in susceptibility to canker

• Resistance to canker is a highly quantitative trait: information about cultivar performance can be conflicting

’Map and control European canker in apple’

• SLF, 2010-2013, 2 100 kkr

Aims:- to identify cultivars with high level of resistance to

the diseasethe disease- to further develop qPCR - based detection method

to be used to detect the patogen Nectria galligenaalready during the period of latent infection

Material

• 58 cvs were tested as cut shoots, 4 shoots/cv in a plastic tent in the greenhouse at Balsgård (2011). Reliable results obtained for 39

• The same 58 cvs were tested as potted trees, 8 trees/cv, in the unheated greenhouse in Alnarp (2011-2012)

• The same 58 cvs were tested under high infection pressure in the field (2011-2012)field (2011-2012)

• 39 cvs were tested as cut shoots in a climate chamber in Alnarp, 8 shoots/cv (2012)

• The same 39 cvs are tested as trees, 12 trees/cv, in the unheated greenhouse in Alnarp

• The same 39 cvs are tested under high infection pressure in the field (2012-2013)

Methods:

Inoculations:• Wound inoculations of cut shoots, 3 inoculations/shoot, 1000

conidia/wound, spring, in a climate chamber, 16 C, 70% RH• Wound inoculations of one-year old trees, 3 inoculations/tree, 1000

conidia/wound, fall-winter and spring, in an unheated greenhouse• ’Natural’ infection under high infection pressure, field• ’Natural’ infection under high infection pressure, field

Assessments: • Colonization rate through measurements of lesions at regular time

intervals; comparisons mellan cultivars are based on AUC• Infection percentage. Comparisons mellan cultivars are based on

ratio of infected leaf scars to the total amount of leaf scars

Results

Aroma, cut shoot, 20 dpi, spring 2011 Elise, tree, 40 dpi, spring 2011Aroma, cut shoot, 20 dpi, spring 2011 Elise, tree, 40 dpi, spring 2011

Cox Orange, tree, 57 dpiSantana, tree, 57 dpi Discovery, tree, 57 dpi

Fall – winter, 2011-2012

Lesion development in one year-old potted trees (mean values were calculated for 8 trees per cultivar)

Infection percentage in one year-old trees (mean values were calculated for 3-4 trees per cultivar)

Differences in resistance Level among 39 apple cvs.Cut shoots, 2011

Differences in resistance Level among 58 apple cvs.Potted trees, 2011-2012

Differences in resistance Level among 39 apple cvs.Cut shoots, 2012

10

15

20

25

0

5

10

Infection percentage under ’natural’ inoculations of one year-old trees

Test of the QPCR primers for their specificity

PCR-products obtained with qPCR primer pairs on the following DNA-samples: 1. N. ditissima grown on apple leaves placed on PDA; 2. Clean culture of N. ditissima grown on PDA; 3. Alternaria solani; 4. In-vitro M26; 5. Infected tissue; 6. Negative control

’Prebreeding for future challenges in Nordic Apples’

WP 3 – Phenotyping of canker susceptibility in applecultivars

• 32 cvs, 8 – common for Balsgård, Graminor and Alnarp (SLF)• Inoculations: 3 shoots/cv, 3 wounds/shoot, 2 inoculation time points, • Inoculations: 3 shoots/cv, 3 wounds/shoot, 2 inoculation time points,

in a plastic tent

Conclusions (methods)

Inoculations on cut shoots

•Visualised the differences between the most susceptible and the most resistant cultivars

• Test can be performed on small greenhouse areas or in a climate chamber and the results can be obtained within 4-6 weeks

• Very useful when there is a need for infected material for other • Very useful when there is a need for infected material for other experiments, e.g. qPCR, microscopy etc

• However, the infection developed very quickly, which requires numerous data recordings, preferably every fifth day

• Different cvs showed different kinds of symptoms which can complicate phenotyping

• The method doesn’t consider possible influence of the rootstock and environment

Inoculations on trees

• Allow observations of the disease and defence reactions and repeated measurements of the lesions under longer time

• It is possible to inoculate trees at different seasons (different state of the trees, different inocula), which results in a better modelling of the situation in orchards situation in orchards

• However, the time (about a year) and investments needed for producing trees limit the amount of cultivars to be tested.

QPCR procedure

• Novel primers from a variable region in the N. galligena genome were developed at our laboratory

• Allows for estimation of the biomass of N. galligena in the presumably infected tissue, but we were not able to detect the fungus in presumably healthy tissue

• However, further improvement of the method is necessary before any conclusions about usefulness of this method as an alternative/complement to phenotyping can be drawn

Conclusions (general)

• Assessment of colonization (AUC) and infection percentage (Inf %) are useful tools for evaluation of resistance to European canker. Cultivars known to be susceptible like Cox Orange and James Grieve had high values for both AUC and Inf %, while the resistant Santana had low AUC and Inf %. and Inf %.

• The relatively susceptible Elise and the relatively resistant Jonathan had low and moderate AUC, respectively, but differed strongly in Inf %. The resistance of these cultivars can be fully understood when considering both components, colonization rate and Inf %. This documents the pruness of Elise to canker infection and supports our previous experience that its cankers do not proceed fast at the early stage of infection.

• For determination of level of resistance in cultivars as well as for dissection of the genetic basis of the resistance, both components of resistance must be distinguished.

• Fall inoculations were more informative than spring inoculations; the fast disease progression in spring demands weekly assessments and produces non-demands weekly assessments and produces non-significant differences among the majority of cultivars.

• Trueness-to-type of all the analyzed cultivars will be verified through SSR genotyping before final conclusions will be made on cultivar susceptibility.

First steps towards genetic dissection of thistraitTwo main components: Colonization rate and Infectionpercentage (phenoping methods available)

Other components (phenotyping methods tobe elaborated):

• Internal spread (assessed by measuring of internal necrosis)

• Proliferation of the pathogen (assessed by quantificationof fungal biomass with qPCR)of fungal biomass with qPCR)

QTL discovery (colonization rate):N

umbe

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seed

ling

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HiDRAS: www://hidras.unimi.itHiDRAS: www://hidras.unimi.it

W.E. van de Weg

Length of cankers (mm, upper value of class)

Lesion length distribution, 133 days after inoculation, high dose of inoculum, indicate limited number of major QTL (van de Weg, unpubl.)

Further research….

• Contribute with our research to common understanding of the genetic background of the resistance and find molecular markers linked to QTLs

• Elucidate molecular mechanisms underlying differences amongcultivars concerning resistance to Neonectria ditissima. Try tocultivars concerning resistance to Neonectria ditissima. Try tounderstand WHAT makes the difference. Candidate genes?

• Find methods to discriminate different isolates (rases???) of Nectriaand identify the infection ways (from nurseries in Belgium and Netherlands, from cankered trees in the orchards???)

OUR CURRENT PROJECT IS A VERY INITIAL STEP….

We, who are involved:

Larisa Gustavsson, Marjan Ghasemkhani, Anna Zborowska (Alnarp)

Hilde Nybom, Jasna Sehic (Balsgård)Hilde Nybom, Jasna Sehic (Balsgård)

Collaborations:CRA-W, Belgium; JKI, Germany; Plant and Food Reseach, New Zealand - phenotyping

WUR, Netherlands – genetic dissection of the trait on a shared population (Jonathan x Prima). Stability of QTL over environments; QTL allele mining in a diversified germplasm.environments; QTL allele mining in a diversified germplasm.

Clemson University, USA – comparison of transcriptomes of parents of maping populations (Jonathan x Prima, NL;Aroma x Discovery, SE; Santana x Aroma (SE), Santana x Katja (SE))

International projects:

’FruitBreedomics’ (coord. by Francois Laurens, INRA)WP3 ’Identification of QTL for fruit quality and disease resistance’ WP4 ’Exploring of phenotypic and genetic diversity in European apple and peachEuropean apple and peach

’NordApp’ – Pre-breeding for future challenges (coord. by Hilde Nybom, SLU)

Thanks!

Cox Orange Liberty