European canker

European canker: a devastating
disease for young apple orchards
in Northern Europe
Larisa Gustavsson
Plant Breeding and Biotechnology
Alnarp
European canker
M. Lateur
• 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 of
hosts
Marc Lateur
Perithecia contain ascospores (genetic
recombination)
Sporodochia contain
conidia (vegetative
reproduction of a fungus)
• 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
Disease cycle of canker caused by Neonectria ditissima
Agrios, 1997
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 xylem
fibers 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
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 Lateur
Walloon Agricultural Research Centre
CRA-W, GEMBLOUX - Belgium
Dept. of Life Sciences
Breeding & Biodiversity Unit
Wounds
after
Plaies
detree
liensties
Wounds after branch
prunings Plaies de taille
= 0,8%
= 13,1%
Aisselles de
Angle of branches
branches =
6,6%
Fruit
Cicatrices
scars
fruitières =
10,1%
Cicatrices
Leaf
scars=
foliaires
69,4%
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
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
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 disease
- to further develop qPCR - based detection method
to be used to detect the patogen Nectria galligena
already 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)
• 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
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
Santana, tree, 57 dpi
Elise, tree, 40 dpi, spring 2011
Discovery, tree, 57 dpi
Fall – winter, 2011-2012
Cox Orange, tree, 57 dpi
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
25
20
15
10
5
0
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 DNAsamples: 1. N. ditissima grown on apple leaves placed on PDA; 2.
Clean culture of N. ditissima grown on PDA; 3. Alternaria solani; 4. Invitro M26; 5. Infected tissue; 6. Negative control
’Prebreeding for future challenges in
Nordic Apples’
WP 3 – Phenotyping of canker susceptibility in apple
cultivars
• 32 cvs, 8 – common for Balsgård, Graminor and Alnarp (SLF)
• 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
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
• 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 %.
• 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 nonsignificant 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 this
trait
Two main components: Colonization rate and Infection
percentage (phenoping methods available)
Other components (phenotyping methods to
be elaborated):
• Internal spread (assessed by measuring of internal
necrosis)
• Proliferation of the pathogen (assessed by quantification
of fungal biomass with qPCR)
Number of seedlings
QTL discovery (colonization rate):
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.)
W.E. van de Weg
HiDRAS: www://hidras.unimi.it
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 among
cultivars concerning resistance to Neonectria ditissima. Try to
understand WHAT makes the difference. Candidate genes?
• Find methods to discriminate different isolates (rases???) of Nectria
and 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)
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.
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 peach
’NordApp’ – Pre-breeding for future challenges (coord. by
Hilde Nybom, SLU)
Thanks!
Cox Orange
Liberty