Assessment of sweet cherry (Prunus avium 
L.) genotypes for response to bacterial 
canker disease
Authors: Josephine Mgbechi-Ezeri, Lyndon Porter,  
Kenneth B. Johnson, and Nnadozie Oraguzie
The final publication is published in Euphytica, available at Springer via 
http://dx.doi.org/10.1007/s10681-017-1930-4.
Mgbechi-Ezeri, Josephine, Lyndon Porter, Kenneth B. Johnson, and Nnadozie Oraguzie. 
"Assessment of sweet cherry (Prunus avium L.) genotypes for response to bacterial canker 
disease." Euphytica 7 (July 2017): 1-12. DOI: 10.1007/s10681-017-1930-4.
Made available through Montana State University’s ScholarWorks 
scholarworks.montana.edu 
Assessment of sweet cherry (Prunus avium L.) genotypes
for response to bacterial canker disease
Josephine Mgbechi-Ezeri . Lyndon Porter . Kenneth B. Johnson .
Nnadozie Oraguzie
Received: 8 October 2016 / Accepted: 21 June 2017 / Published online: 24 June 2017
 Springer Science+Business Media B.V. 2017
Abstract Integration of alleles for bacterial canker
resistance into new sweet cherry cultivars requires
information on the sources of resistance in the
germplasm. Five market-leading sweet cherry culti-
vars, ‘Rainier’, ‘Sweetheart’, ‘Bing’, ‘Regina’ and
‘Chelan’, advanced selections ‘AA’, ‘BB’, ‘CC’,
‘DD’, ‘EE’, ‘GG’, and ‘PMR-1’ used as breeding
parents in the Washington State University’s Sweet
Cherry Breeding Program were evaluated. Compara-
tive genotypic disease severity was obtained with
three methods of inoculation (leaf wounding with
carborundum, cut wounds in leaf mid-rib and shoot
tip) on whole plants. Additionally, genotypic data on
susceptibility of detached leaves versus fruit and an
assessment of the movement of Pseudomonas syr-
ingae pv. syringae (Pss) population in inoculated
shoots were obtained. Genotype susceptibility was
significantly (P B 0.05) influenced by inoculation
method, with shoot inoculation providing the best
separation of resistance levels among genotypes. A
low correlation (r = 0.26, P = 0.21) was observed
between disease responses measured on detached leaf
versus fruit, while a moderately high correlation
(r = 0.50, P = 0.10) was found among bacterial
populations in the tissues and in the degree of
symptoms expressed. By all comparative methods,
the advanced selections, as well as, ‘PMR-1’, were
less susceptible than the market-leading cultivars.
Also, movement of Pss from shoot tip inoculation
points to the shoot base was not detected for advanced
selections ‘AA’, ‘BB’, ‘DD’, and ‘EE’. This study
reveals that the advanced selections could be potential
sources of resistance alleles to bacterial canker. This is
the first evaluation of the advanced selections for
bacterial canker disease.
Keywords Allele  Bacterial canker  Disease
resistance  Sweet cherry  Pseudomonas syringae pv.
syringae
Introduction
Bacterial canker, caused by Pseudomonas syringae
pv. syringae (Pss) and Pseudomonas syringae pv.
morsprunorum (Psm), is one of the economically
J. Mgbechi-Ezeri (&)
Department of Plant Science and Plant Pathology,
Montana State University, Bozeman, MT, USA
e-mail: josephi.mgbechiezeri@montana.edu
L. Porter
Grain Legume Genetics and Physiology Research Unit,
USDA-ARS, Prosser, WA, USA
K. B. Johnson
Department of Botany and Pathology, Oregon State
University, Corvallis, OR, USA
N. Oraguzie
Department of Horticulture, Washington State University-
Irrigated Agriculture Research and Extension Center,
Prosser, WA, USA
important diseases of sweet cherry and other stone
fruits worldwide (Crosse 1966; Kennelly et al. 2007;
Renick et al. 2008; Vicente et al. 2004). While in some
orchards both pathovars can cause bacterial canker
symptoms and co-exist, strains of Pss are more
virulent on sweet cherry (Kennelly et al. 2007). The
pathogen attacks various parts of the tree and the
disease is recognized by a variety of symptoms
depending upon the part of the plant being attacked
(Moore 1988). These symptoms include trunk and
branch cankers, gummosis, blossom blast, twig blight,
shoot dieback and leaf shot hole (Moore 1988).
Moreover, the pathogen has a wide host range and
lives as an epiphyte on numerous plants without
producing symptoms. For susceptible cultivars, the
ubiquitous nature of Pss makes it potentially destruc-
tive whenever favorable environmental conditions
occur (Cameron 1970; Kennelly et al. 2007). These
conditions include prolonged wetness and frost-in-
duced wounding (Moore 1988; Otta and English 1970;
Sobiczewski and Jones 1992). In the USA, losses of
sweet cherry trees to bacterial canker have approached
75% in Oregon (Cameron 1962) and Michigan
(Renick et al. 2008).
Management of bacterial canker in susceptible
sweet cherry cultivars is frequently unachievable due
to lack of effective chemical control and the epiphytic
nature of the pathogen (Kennelly et al. 2007). Like-
wise, cultural control practices such as removal of
infected twigs/trees, summer pruning and soil amend-
ment can only reduce symptoms but does not eliminate
populations of the pathogen (Spotts et al. 2010a).
Conversely, the use of resistant scion and rootstock
cultivars holds promise as a sustainable and cost
effective method of bacterial canker control. But, it is
also frustrating due to limited availability of resistant
cutivars and challenges with screening methods to
distinguish levels of resistance within the breeding
population. Previously, various methodologies have
been employed to assess the resistance of sweet cherry
scion and rootstock cultivars to Pss. Bedford et al.
(2003) evaluated two inoculation methods (leaf disc
infiltration and mid-rib wound injection) in a detached
leaf assay to differentiate disease response among
sweet cherry cultivars. This study revealed failure of
leaf disc infiltration to discriminate disease response
among cultivars. Spotts et al. (2010b) who compared
the resistance of five sweet cherry cultivars based on
seven inoculation methods showed that susceptibility
of sweet cherry cultivars was significantly influenced
by inoculation method, and that cvs. ‘Regina’ and
‘Rainier’ were tolerant to Pss infection. This was
contrary to Roche (2001) who observed that ‘Rainier’
was highly susceptible to Pss based on an excised twig
assay. Garrett (1986), in an investigation of the
influence of rootstock on susceptibility of sweet
cherry reported variation in susceptibility of cultivars
based on methods of inoculation. Overall, these past
studies demonstrate variability when attempting to
characterize the tolerance/resistance and susceptibility
of sweet cherry selections to bacterial canker. This
variability likely stems from inoculation method,
pathogen strain and type of plant materials utilized
for experiments as well as the environment.
Development of new sweet cherry cultivars is an
ongoing activity. Consequently, continuous assess-
ment of disease resistance in breeding germplasm is
necessary to maintain and enhance the utility, dura-
bility and acceptance of new cultivar releases. Previ-
ous studies have evaluated resistance of some sweet
cherry cultivars to economically important diseases
including powdery mildew and bacterial canker.
Toyama et al. (1993) found that the sweet cherry
cultivar ‘PMR-1’, originating from an open-pollinated
seedling, was resistant to powdery mildew disease. In
2001, Olmstead et al. conducted a study on the
inheritance of powdery mildew resistance among
progenies from biparental crosses and their reciprocals
including sweet cherry cultivars, ‘PMR-1’ 9 ‘Bing’/
‘Rainier’/ and ‘Van’. Subsequently, Zhao (unpub-
lished) concluded that genotypes including ‘AA’,
‘BB’, ‘CC’, ‘DD’, ‘EE’ and ‘GG’ from Olmstead
et al.’s study were resistant to powdery mildew
disease, which, combined with average fruit quality,
elevated these genotypes to advanced selection status
in the Washington State University breeding program.
Moreover, in an evaluation of 600 sweet cherry
genotypes, selections ‘AA’, ‘BB’, ‘CC’, ‘DD’, ‘EE’
and ‘GG’ also showed a low response to bacterial
canker disease based on a mid-rib inoculation in a
detached leaf assay (Mgbechi-Ezeri 2016). To date, no
other studies have evaluated these selections for
resistance to bacterial canker. For the current study,
we collected comparative genotypic data on suscep-
tibility to bacterial canker based on whole plant
inoculation of Pss into three types of wounds: leaf
wounds created with carborundum, cut leaf mid-ribs,
and cut shoot tips. In addition, we compared disease
responses among detached leaves and detached fruit
following inoculation, and correlated the size of the
pathogen population in the stem tissue with degree of
symptom expression. The overarching goal of the
study was to develop methods to accurately identify
sweet cherry genotypes resistant to bacterial canker
for use as parents in the breeding of new sweet cherry
cultivars for the Pacific Northwest regions of United
State of America.
Materials and methods
Plant materials
Six advanced selections ‘AA’, ‘BB’, ‘CC’, ‘DD’,
’EE’, ‘GG’ (from‘PMR-1’ 9 ‘Rainier’ or the recip-
rocal cross) (Olmstead et al. 2001) and their parent,
‘PMR-1’, were chosen for this study based on their
low disease response to bacterial canker in a previous
detached leaf assay (Mgbechi-Ezeri 2016). Also,
chosen for evaluation were five market-leading culti-
vars (‘Regina’, ‘Sweetheart’, ‘Rainier’, ‘Bing’ and
‘Chelan’), which are known to be moderately to highly
susceptible to bacterial canker (Bedford et al. 2003;
Mgbechi-Ezeri et al. 2013; Spotts et al. 2010b). All
plant materials originated from the United States, and
are maintained by the Washington State University
sweet cherry breeding program (WSU-SCBP) located
at Irrigated Agriculture Research and Extension
Center, Prosser, WA. Based on previous studies, the
advanced selections and ‘PMR-1’ have been identified
as sources of powdery mildew resistance and used as
parents for powdery mildew resistant breeding in the
WSU-SCBP (Toyama et al. 1993; Zhao unpublished).
For this study, scions of all genotypes were budded
onto ‘Mazzard’ rootstocks (14.3 mm thickness) that
had been propagated by a commercial nursery and
received as dormant bare rooted trees covered with
wood shavings. Budded rootstocks (four buds per
rootstock and two plants per genotype) were trans-
planted into 15-l plastic pots containing Nulife
ProBlend potting soil (Waupaca, WI, USA) and grown
under 12 h of light (fluorescent bulb) in the green-
house. Trees were budded in early January (2014 and
2015) for a spring inoculation experiment and again in
early November (2014) for a winter inoculation
experiment. After budding, trees were maintained in
the greenhouse at a temperature between 22 and 25 C
and allowed to grow for approximately 6 weeks before
inoculation. Budded rootstocks were watered regu-
larly as needed, and fertilized after bud break (using
Miracle Gro All-purpose plant food, 24-8-16, Scots
Co., Marysville, OH) until the experiment was
concluded.
Bacterial strain and inoculum preparation
Pseudomonas syringae pv. syringae, Pss 425, iso-
lated previously from a diseased sweet cherry tree
located near the Dalles, OR, as was obtained from
Department of Botany and Plant Pathology, Oregon
State University, Corvallis. Pss 425 was further
characterized with LOPAT (Levan production, oxi-
dase activity, potato soft rot, arginine dihydrolase
activity and hypersensitivity on tobacco) and GATTa
(gelatin liquefaction, aesculin hydrolysis, tyrosinase
activity and utilization of tartrate) tests (Lelliott and
Stead 1987), and confirmed for syringomycin produc-
tion by polymerase chain reaction (PCR) with primer
pair PSSB1 and PSSB2 (Sorensen et al. 1998). This
bacterial strain was chosen due to its high virulence
compared to five other Pss strain in preliminary
inoculation of several sweet cherry cultivars (Mg-
bechi-Ezeri unpublished). A loopful (10 ll) of bacte-
rial culture grown for 48 h on a Pseudomonas Agar F
(PAF, Difco Labs, Detroit, MI) amended with 50 lg/
ml cycloheximide (Sigma-Aldrich C7698-5G) was
transferred to a sterile nutrient broth (15 ml) and
agitated on a rotary shaker for 24 h at 200 rpm and
28 C. Bacterial cells were harvested by centrifuga-
tion at 7000 rpm for 5 min, re-suspended in sterile
distilled water (SDW) and adjusted to cell densities of
108 cfu/ml (absorbance at 600 nm = 0.3). Bacterial
cell counts were confirmed by dilution plate method
on a PAF medium and then 0.5% of surfactant (Break-
Thru S 240, Evonik Industries, Essen, Germany) was
added to the inoculum before inoculation. SDW mixed
with 0.5% surfactant was used as a negative control
except for inoculation by carborundum where 0.05 g
carborundum powder plus SDW was used.
Experiment 1: comparison of inoculation methods
In the greenhouse, disease responses of 12 sweet
cherry genotypes were compared in whole plant assays
after inoculation of Pss into three types of wounds: leaf
wounds created with carborundum, cut leaf mid-ribs,
and cut shoot tips.
Carborundum inoculation
The first, second and third leaves from the apical
growing point of the shoot of each genotype were
surface sterilized with 70% ethanol for about 30 s and
rinsed with SDW. The leaves were inoculated by
rubbing a sterile cotton swab (Q-tips cotton swab,
Unilever, USA) dipped in a 108 cfu/ml suspension of
Pss 425 mixed with 0.05 g carborundum powder (320
grit, Alfa Aesar , Johnson Matthey, Ward Hill, MA)
on the abaxial side of the leaf from the distal to
proximal edges twice. Water plus 0.05 g carborundum
was used as a negative control. Inoculated leaves were
enclosed with a plastic bag containing sterile cotton
balls soaked in SDW for 48 h to create a 100% relative
humidity environment suitable for the pathogen to
initiate infection. Disease severity was recorded at
8 days post inoculation (dpi) based on a 5-point scale
adapted from Bedford et al. (2003) (Table 1).
Mid-rib inoculation
The first, second and third leaves from the apical
growing point of the shoot of each genotype were
surface sterilized as described above. The leaves were
inoculated by carefully slitting the mid-rib to about
1 mm in depth from the petiole to the apex on the
underside of the leaf using a sterile scalpel (blade size
11, Bard-Parker ref: 371311) and applying 10 ll of the
Pss 425 suspension (108 cfu/ml) to the wounded area.
On control plants, leaves were inoculated with an
equal volume of sterile distilled water. The inoculated
leaves were enclosed within plastic bags containing
sterile cotton balls as described above. Disease
severity was recorded at 8 dpi based on a 5-point
scale (Table 1).
Shoot inoculation
Shoots were inoculated by cutting the tip about 20 mm
in length with a sterile razor blade to which 10 ll of
Pss 425 (108 cfu/ml) was applied directly on top of the
wound. Wounds were covered with plastic bags
containing sterile cotton balls as described above.
The cotton balls were prevented from contact with the
inoculation site. Necrotic lesion length was measured
after 8 weeks, and disease severity was rated on a
5-point scale (Table 1) (Santi et al. 2004).
The wounding experiment was arranged in com-
pletely randomized experimental design with five
replicates of two plants per genotype. Experimental
trees were spaced 300 mm between pots and main-
tained in the greenhouse at 15 C. Each method of
inoculation was applied to one of the grafted shoots on
each tree; the shoot that emerged from the fourth bud
was used as a negative control. The experiment was
conducted three times: spring 2014, winter 2014 and
spring 2015.
Experiment 2: correlation between detached fruit
and foliar disease responses
The protocol of Latorre et al. (2002) was used for
inoculation of detached immature fruit. In May, 10
immature fruit (light green stage) from each of the
Table 1 Rating scale and disease response categorization of sweet cherry genotypes to infection by Pseudomonas syringae pv.
syringae
Rating
scale
Greenhouse rating (Bedford et al.
2003)
Laboratory rating (Roche 2001). Fruit rating (Moragrega et al. 2003) Disease
category
0 Free from infection No necrosis No necrosis Resistant
1 1–25% of shoot infected Necrosis restricted to line of
infection
Necrosis restricted to the point of
infection
Low
2 C26 to B50% of shoot infected Necrosis B50% of leaf area Necrotic area\5 mm diameter on
fruit
Moderate
3 C50 to B75% of the shoot Necrosis[50% of leaf area Necrotic area 5–10 mm diameter on
fruit
High
4 [75% of the shoot infected/death
of tree
Total necrosis of leaf Necrotic area[10 mm diameter on
fruit
High
twelve genotypes were detached from fruit-bearing
trees planted at the WSU Roza Experimental Orchard
Prosser, WA. (All scion genotypes were grown on a
‘Gisela 6’ rootstock except ‘Chelan’ and ‘PMR-1’,
which were on a ‘Mazzard’ rootstock). The fruits were
surface sterilized with 70% ethanol for 30 s, rinsed in
two changes of SDW and blotted dry with three layers
of sterilized paper towels. Each fruit was punctured
with a sterile scapel blade size 11 to a depth of about
1 mm, and inoculated with 10 ll of 108 cfu/ml
bacterial suspension. Following inoculation, fruit were
placed in plastic boxes containing three layers of moist
sterile paper towel and incubated at room temperature
for 8 days. Control fruit of each genotype were
inoculated with SDW. The experiment was arranged
in a completely randomized design with three repli-
cates per genotype; the experiment was conducted in
2014 and repeated in 2015. Disease symptom expres-
sion and progression was scored using a 5-point scale
(Moragrega et al. 2003) (Table 1) at 8 dpi. To compare
disease responses from fruit to foliar responses, 10
new leaves from the tip of each genotype were
collected from the orchard along with the immature
fruit. Detatched leaves were inoculated with Pss 425
following the cut mid-rib protocol as described above.
After inoculation, these leaves were incubated in
plastic boxes lined with moistened paper towel as
described above, and scored for disease response at 8
dpi.
Experiment 3: correlation between pathogen
population size in tissue and symptom expression
At 8 weeks after inoculation, 200 mm lengths of
sweet cherry shoots that had been inoculated with Pss
425 following the shoot tip protocol (described above)
were harvested. On each shoot, a 10 mm
length 9 2 mm stem thickness piece of tissue was
sampled from three areas of the stem. The positions
were labeled as top (area circumscribing infection),
middle (area between the infected region and the base
of the shoot) and base (area located at the base of the
shoot with no visible symptom). Removed tissue was
placed in Ziploc bags and kept on ice in a cooler for
transport to the laboratory and processed immediately.
Tissue from non-inoculated shoots also was sampled
similarly to represent a negative control. Each tissue
piece (approximately 0.1 g) was soaked in 1 ml of
SDW for about 6–8 h at room temperature, tenfold
dilutions were prepared and 1 ml aliquots of 106
dilution (based on preliminary study) was spread on
PAF plates amended with 50 lg/ml cycloheximide in
three replicates per sample. The plates were incubated
at 28 C and colonies counted after 72 h to calculate
the CFU/gram of infected tissue. A 4-point rating scale
(0–3) was applied to describe bacterial advancement
where, 0 = no bacterial cells detected, 1 = bacterial
cells detected at the top, 2 = bacterial cells detected
mid-way, and 3 = bacterial cells detected at the base.
Statistical analysis
All experiments were analyzed by analysis of variance
(ANOVA) in SAS 9.4 (SAS Institute Inc., Cary, NC,
USA) (2014). A Bartlett’s test was used to test for
homogeneity of variances before combining the data
from different years (e.g., spring 2014, winter 2014
and spring 2015). ANOVA was used to examine the
disease response of each genotype and how the
inoculation methods differentiated disease responses
among genotypes. The means were separated by
Tukey’s Studentized Range (HSD) test (P = 0.05).
Pearson’s correlation coefficient was used to compare
disease responses among detached fruits and detached
leaves, as well as, to compare pathogen advancement
in stem tissue with severity of symptom expression.
Pathogen population sizes, was estimated using the
formula CFU/g = number of colonies counted 9 di-
lution factor /volume of culture plated. The resulting
colony forming unit was log transformed to get the
Log CFU then analyzed by ANOVA.
Results
Bacterial strain
The bacterial strain used in the experiments, Pss 425,
produced levan, induced hypersensitive reaction on
tobacco leaves, hydrolyzed gelatin and aesculin but
was negative for oxidase, arginine dihydrolase, potato
soft rot, tyrosinase activity and utilization of tartaric
acid (?—? reaction for LOPAT and ??—for
GATTa test) (Lelliot et al. 1966; Schaad et al. 2001).
A PCR product size of 752 bp specific for the syrB
gene encoding syringiomycin secretion was amplified.
Overall, these results confirmed the identification of
the bacterial strain as Pss.
Comparison of inoculation methods
Disease response among genotypes differed signifi-
cantly (P B 0.05) by inoculation method, although all
the plants challenged with Pss 425 were infected
regardless of inoculation protocol (Fig. 1). A brown
discoloration was observed on negative controls at the
point(s) of wounding but did not progress beyond the
injury site. Homogeneity of variance (evaluated with a
Bartlett’s test) revealed no significant differences in
overall disease response among experiments per-
formed at different times (mid-rib: P = 0.33; car-
borundum; P = 0.2 and shoot; P = 0.25). Initial
necrotic symptoms were observed on all genotypes
at 2, 4 and 14 dpi for mid-rib, carborundum and shoot
inoculations, respectively. All genotypes inoculated
by carborundum had low disease scores that ranged
from 0.9 and 1.3 (Fig. 2); although genotype was a
significant influence on measured values (P B 0.05).
In contrast, disease scores for mid-rib inoculation
ranged between 1.7 and 3.1, potentially indicating a
greater differentiation of resistance and susceptibility
to bacterial canker (Fig. 2). The disease rating of
‘Rainier’ and ‘Sweetheart’ were significantly
(P B 0.05) higher than those for ‘Bing’ and ‘Chelan’,
whereas ‘Regina’, ‘PMR-1’, as well as, the advanced
selections were not significantly different (P C 0.05)
from each other (Fig. 2). With shoot inoculation, the
lesion length ranged between 18.05 and 58.54 mm.
Disease progression and severity were significantly
(P B 0.05) higher in ‘Sweetheart’ ‘Rainier’ and
‘Bing’ than in ‘Chelan’ and ‘Regina’, while the
advanced selections were the most resistant (Fig. 3).
Although the advanced selections showed no differ-
ences in disease response amongst themselves, they
did show reduced infection compared to the market-
leading cultivars and to their parent ‘PMR-1’.
Correlation between detached fruit and foliar
disease response
Severity of symptom on sweet cherry genotypes after
inoculation with Pss differed among fruit and leaves.
The average disease score for immature fruit was
significantly greater (P B 0.05) than in the leaves
(Fig. 4), and none of the inoculated fruit were scored
as resistant. Analysis of variance showed no signifi-
cant differences (P C 0.05) in the effect of year or
genotype 9 year interaction on the disease response
of both fruit and leaf (Table 2). For specific genotypes,
Fig. 1 Necrotic symptoms observed on sweet cherry genotypes
inoculated with Pseudomonas syringae pv. syringae strain Pss
425. a Leaf symptom 8 days after inoculation using
carborundum inoculation, b leaf symptom 8 days after inocula-
tion using mid-rib inoculation, and c shoot symptoms 8 weeks
after inoculation using shoot tip wounding method
‘Regina’ had the highest disease scores (3.2 and 1.3 for
fruit and leaf, respectively) while ‘EE’ had the lowest
score (2.3 and 1.1, respectively). Genotypic scores for
the detached leaf and fruit assays were only weakly
correlated (r = 0.26, P = 0.21).
Correlation between bacterial population density
and symptom expression
Pathogen population size within diseased shoots
varied among genotypes and position of the sample
taken from the inoculated shoot. Overall, population
size of Pss 425 was reduced significantly (P B 0.05) in
advanced selections compared to market-leading
cultivars (Table 3). At the shoot tip (site of inocula-
tion), pathogen population size differed significantly
(P B 0.05) among advanced selections and ‘PMR-1’.
‘EE’ had a relatively low pathogen population (log
(cfu/g) = 7.59 ± 0.02) while ‘AA’ had the highest
(log (cfu/g) = 8.18 ± 0.01) pathogen population.
Among the market-leading cultivars, pathogen popu-
lation size did not vary significantly (P C 0.05) with
‘Chelan’ having the lowest population size (Table 3).
At this sampling position, the overall pathogen
population was relatively high in all genotypes
challenged with Pss.
For stem tissue collected from the middle of the
diseased shoots, internal discoloration of the vascular
Fig. 2 Comparison of mid-rib and carborundum methods of
inoculation with Pseudomonas syringae pv. syringae strain Pss
425 based on disease ratings of 12 sweet cherry genotypes to
bacterial canker. Bars labelled with the same letters within
inoculation methods are not significantly different based on
Tukey’s Studentized Range Test (HSD). Disease severity was
rated 8 days post inoculation. The experiment was conducted
three times (spring 2014, winter 2014 and spring 2015), and the
means were combined across experiments due to lack of
significant differences (P C 0.05) between genotypic values
across experiments
Fig. 3 Length of necrotic lesions on shoots of 12 sweet cherry
genotypes inoculated with Pseudomonas syringae pv. syringae
strain Pss 425 and observed at 8 weeks after inoculation. Bars
labeled with the same letters are not significantly different based
on Tukey’s Studentized Range (HSD) Test at P = 0.05. The
experiment was conducted three times (spring 2014, winter
2014 and spring 2015), and the means were combined across
experiments due to lack of significant differences (P C 0.05)
among genotypic values across experiments
tissue was observed in most genotypes. Pathogen
population size varied significantly (P B 0.05) among
the advanced selections and ‘PMR-1’ although this
was greatly (P B 0.05) reduced compared to the
market-leading cultivars with no substantial (P C
0.05) variation in pathogen population size. At the
base of the shoots, no visual symptom of infection was
observed during sampling. Also, bacterial cells were
not detected in advanced selections including ‘AA’,
‘BB’, ‘DD’ and ‘EE’, while, among the market-
leading cultivars there was no significant variation
(P C 0.05) in the population of Pss . A moderately
high correlation (r = 0.50, P = 0.10) was observed
between pathogen population size and lesion length in
the genotypes.
Discussion
In our earlier study (Mgbechi-Ezeri 2016), use of a
detached leaf assay to screen a large populations of
sweet cherry genotypes for disease response to
bacterial canker showed that the selections ‘AA’,
Fig. 4 Comparison of detached fruit and foliar disease
response under laboratory conditions after inoculation of sweet
cherry genotypes with Pseudomonas syringae pv. syringae
strain Pss 425. Prior to inoculation, leaves were wounded by
creating a cut of about 1 mm in depth in the mid-rib and fruit
were wounded with a puncture of about 1 mm-depth into the
flesh. Both tissues were inoculated with 10 ll of 108 cfu/ml
bacterial suspensions and incubated on a moist sterile paper
towel. Disease response was scored 8 days after inoculation.
The experiment was repeated twice. Since no significant
differences (P C 0.05) were found between values across
experiments using a T-test, a single mean value was obtained
per tissue type and compared
Table 2 Analysis of variance (ANOVA) of the disease
response of 12 sweet cherry genotypes inoculated with Pseu-
domonas syringae pv. syringae strain Pss 425 in 2 years (2014
and 2015) and in two different tissues (leaf and fruit) in a
detached assay
Source Mean square
DF Leaf Fruit
Genotype 11 6.17*** 0.5***
Year 1 0.02 0.13
Genotype 9 year 11 0.17 0.03
Error 24 0.13 0.04
Experiment was conducted in the laboratory with detached
fruit (light green stage) and leaves collected from the same tree
in the field. Disease was scored on a 0–4 scale
*** Significant at P B 0.0001
Table 3 Population size of Pseudomonas syringae pv. sy-
ringae strain Pss 425 at different shoot locations on inoculated
shoots from 12 sweet cherry genotypes on a ‘Mazzard’
rootstock
Genotypes Sourceb Sampling locationa
Topc Middle Based
Bing WSU-SCBP 7.90 c 7.78 a 7.19 a
Chelan WSU-SCBP 7.81 d 7.59 ab 6.70 ab
PMR-1 WSU-SCBP 7.71 e 7.13 cd 6.73 ab
Rainier WSU-SCBP 7.93 c 7.85 a 6.56 ab
Regina WSU-SCBP 7.91 c 7.78 a 7.11 ab
Sweetheart WSU-SCBP 7.90 c 7.84 a 7.30 a
AA WSU-SCBP 8.18 a 6.10 g ND c
BB WSU-SCBP 7.74 de 6.84 def ND c
CC WSU-SCBP 7.70 e 6.49 fg 4.10 b
DD WSU-SCBP 8.09 b 6.59 ef ND c
EE WSU-SCBP 7.59 f 6.96 cde ND c
GG WSU-SCBP 7.75 de 7.30 bc 6.69 ab
a Sampling location on shoot where top means tissue samples
collected from the shoot tip region circumscribing necrotic
lesions, middle indicates samples collected half way between
the infected shoot tip and the base while base means the base of
the shoot. Total shoot length was approximately 200 mm
b WSU-SCBP, Washington State University sweet cherry
breeding program. All plant materials originated from the USA
c Bacterial count in cfu/g of infected stem tissue transformed
into log (cfu/g). Values followed by the same letters within
column are not significantly different at P B 0.05 based on
Tukey’s Studentized Range Test (HSD). Bacterial populations
were determined using the dilution plate count method on PAF
plates amended with 50 lg/ml cycloheximide and incubated at
28 C for 72 h in three replicates
d ND indicates bacterial cell was not detected at a dilution of
1 9 106 (dectection limit)
‘BB’, ‘CC’, ‘DD’, ‘EE’, ‘GG’ as well as, ‘PMR-1’ had
a low disease response. In this study, we used whole
plant assays to determine the disease response of these
advanced selections and important market cultivars of
sweet cherry. Three inoculation methods were eval-
uated to determine the response of 12 genotypes to
infection. Disease symptoms including leaf necrosis
(mid-rib and carborundum) and shoot dieback were
observed on all inoculated plants; disease response
however, varied by genotype and inoculation method.
All genotypes inoculated by carborundum showed
relatively low levels of disease indicating that this
method may not be suitable for differentiating geno-
types for susceptibility or resistance to infection. This
result is in line with the inability of carborundum
inoculation to induce bacterial blight symptoms in
cotton genotypes following inoculation with Xan-
thomonas axonopodis (Eddin et al. 2005). With leaf
mid-rib inoculation, the distinction in the level of
disease response observed among genotypes was
improved compared to wounding with carborundum.
The disease scores in the advanced selections includ-
ing ‘AA’, ‘BB’, ‘CC’, ‘DD’, ‘EE’, ‘GG’ as well as,
‘PMR-1’ were significantly lower than in the cultivars
except for ‘Regina’. The cultivars ‘Rainier’, and
‘Sweetheart’ were more susceptible to bacterial
canker compared to ‘Bing’ and ‘Chelan’ This result
agrees with previous findings where cv. ‘Rainier’,
‘Sweetheart’ and ‘Bing’ were susceptible to bacterial
canker in detached leaf assays in the laboratory
(Bedford et al. 2003; Mgbechi-Ezeri et al. 2013;
Roche 2001). Compared to mid-rib and carborundum
inoculations, the shoot inoculation method resulted in
the greatest differentiation of disease response among
the advanced selections and their parent, ‘PMR-1’, as
well as, among the common market cultivars. Conse-
quently, we conclude that this method will be most
suitable for validating the resistance or susceptibility
of breeding materials.
The economic importance of a disease depends on
the rate at which it progresses on the plant and limits
yield (Keane and Kerr 1997). Identifying an evalua-
tion method that can differentiate the rate of disease
progression among sweet cherry genotypes for poten-
tial use as breeding parents is essential in breeding
programs. This study showed that disease progression
and severity varied among the twelve genotypes
evaluated. For example, ‘Sweetheart’, ‘Rainier’ and
‘Bing’ were more susceptible to Pss than other
cultivars. This concurs with Bedford et al. (2003),
Mgbechi-Ezeri et al. (2013) and Roche (2001) who
had previously reported that ‘Rainier’ was susceptible
in detached leaf and twig assays, while (Spotts et al.
2010b) demonstrated that ‘Rainier’ was tolerant in an
orchard experiment. A likely reason for the discrep-
ancy in reports may be the environment where studies
were conducted and/or the bacterial strain used in the
studies. In detached leaf assays or in a greenhouse
environment, relative humidity is usually higher but
the environment is much more controlled than in
orchard environments. Disease progression under high
humidity is likely to be greater than in a dry
environment as the field (Latorre et al. 1985). In all
inoculation methods, ‘Regina’ showed a low disease
response to Pss, a finding that agrees with (Spotts et al.
2010b). Disease progression on ‘Chelan’ varied with
inoculation method. This suggests that under favor-
able conditions, ‘Chelan’ may display susceptibility to
Pss. All the advanced selections plus ‘PMR-1’
consistently showed low disease progression in all
three inoculation methods suggesting that these geno-
types may have some level of resistance hence may be
potential parents for bacterial canker resistance breed-
ing. These results concur with our previous laboratory
screening based on a detached leaf assay (Mgbechi-
Ezeri 2016). Further evaluation in the field however,
under different environmental conditions may be
necessary to confirm their resistant status.
Other studies have suggested that the contrasting
response of fruit and leaf to infection may be due to
different genetic factors controlling disease reaction in
these tissues (Walker and Bosland 1999; Werner et al.
1986). In this study, we observed significant differ-
ences in disease response between immature fruit and
leaf tissues. One possible explanation for this differ-
ence could be the moisture level and amount of
substrate available in the fruit supporting rapid
bacterial growth, which may be lacking in the leaf.
The result concurs with Werner et al. (1986) who
showed a low correlation between the fruit and leaf to
bacterial canker infection in peach and nectarine. In
addition, significant differences in disease response
were observed between different parts of tomato
plants infected by Phytohpthora capsici (Quesada-
Ocampo and Hausbeck 2010). It may be essential to
incorporate disease resistance alleles from both tissue
types into new cultivars rather than from one tissue
type alone.
Two basic ideas proposed to guide the description
of plant disease resistance include: (1) visual symptom
is a direct consequence of the amount of pathogen
present in a host, and (2) pathogen growth and
development is interrelated with the host’s defense
mechanism and the host’s ability to recognize the
pathogen and activate the defense genes (Kover and
Schaal 2002). Some past studies have shown discrep-
ancies in the findings for the relationship between
symptom expression and bacterial population size. For
example, Vanneste et al. (2013) reported a low
correlation between endophytic population size of
Pseudomonas syringae pv. actinidiae (Psa) and length
of necrosis while Shane and Baumer (1987) docu-
mented a high positive correlation between symptom
expression and population size of Pseudomonas
syringae pv. syringae in wheat. In our study, we
observed a moderatetly high correlation between
visual symptom expression and bacterial population
size. This suggests that applying visual assessments of
necrotic symptoms alone in the evaluation of geno-
types for breeding may not be very efficient for
selecting bacterial canker resistant genotypes. The
ability to detect bacterial cells in some genotypes at a
region farther from the point of inoculation (although
disease score was low) indicates that such genotypes
may be susceptible to infection but can suppress or
delay the effect of the pathogen on the plant. Such
genotypes may be considered partially resistant since
they show minimal damage despite substantial patho-
gen level. The bacterial populations in the advanced
selections and cultivars were high at the region
circumscribing infection, however, moving farther
down to the base of the shoot; it appears that host
defenses in the advanced selections could inhibit the
movement of the bacterial cells through the shoot
tissue unlike in the cultivars. This suggests that
although the advanced selections showed necrotic
symptoms after inoculation with Pss, they still had
some level of resistance not present in the cultivars and
thus, could be considered as sources of resistance to
Pss in the sweet cherry breeding program. Previous
studies (Mgbechi-Ezeri et al. 2013; Spotts et al.
2010b) reported that ‘Regina’ shows resistance to
bacterial canker as demonstrated by visual expressions
of necrotic symptoms in the field, greenhouse and in a
detached leaf assay. In this study, we also found that
the advancement of P. syringae pv. syringae in
‘Regina’ when compared with other known
susceptible cultivars did not differ significantly in all
the positions examined. In three environments (field,
greenhouse and laboratory) and in different experi-
ments and time periods, ‘Regina’ consistently dis-
played a low disease response. Moreover, despite the
high level of pathogen populations in ‘Regina’ in
positions farther away from the point of inoculation,
the low disease response of ‘Regina’ was still evident.
This indicates that ‘Regina’ may exhibit some level of
resistance. More research is needed to determine if
predisposing factors such as poor soil conditions, frost
damage and high humidity may influence the disease
response of ‘Regina’.
Conclusion
This study has demonstrated that the disease
severity in the sweet cherry cultivars including
‘Rainier’, ‘Sweetheart’ and ‘Bing’ was higher
compared to ‘Chelan’, and ‘Regina’. Based on the
distinctive discrimination in disease response
among genotypes with shoot inoculation, this
method will be preferred for evaluation of sweet
cherry genotypes for resistance to bacterial canker
disease. All the advanced selections evaluated
showed low disease response with ‘AA’, ‘BB’,
‘DD’ and ‘EE’ exhibiting some form of resistance
by inhibiting the migration of bacterial cells from
the point of inoculation to the base of the shoot.
This result demonstrates that the advanced selec-
tions could be potential sources of allele donors for
resistance to bacterial canker. The low correlation
between fruit and foliar disease symptom expres-
sion is an indication that different genetic factors
may be influencing bacterial canker in the fruit and
other plant tissues. Pyramiding resistance alleles
from both leaf and fruit tissues in new cultivars will
be essential for effective disease control. Using
visual disease rating alone may not provide suffi-
cient information to fully determine the level of
disease resistance in an individual genotype. Other
parameters such as quantifying the pathogen pop-
ulation and level of migration from the point of
inoculation may also be important.
Acknowledgements We thank Noel Munoz for tree budding
and Mojtaba Chavoshi and Sue Watskin for assistance with
greenhouse experimentation. This work was funded in part from
royalty income from the sweet cherry breeding program.
Compliance with ethical standards
Conflict of interest The authors declare that they have no
conflict of interest.
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