Otto2018 Article BacterialCankerOfCherryTreesPr
Otto2018 Article BacterialCankerOfCherryTreesPr
Otto2018 Article BacterialCankerOfCherryTreesPr
https://doi.org/10.1007/s10658-017-1384-5
Abstract In the 1980’s the causal agents of bacterial study showed a hypersensitive response on tobacco
canker of cherry trees in South Africa was reported to be leaves and were pathogenic on immature cherry fruit
Pseudomonas syringae pv. syringae and Pseudomonas and cherry trees. The phenotypic tests and MLSA using
syringae pv. morsprunorum. Subsequently, no further four genes (cts, gapA, gyrB and rpoD) showed pheno-
studies were undertaken on the disease or causal agents. typic and genetic identity with Pseudomonas syringae
The aim of the current study was to conduct field sur- pv. syringae. Selected strains induced a hypersensitive
veys to determine the current situation pertaining to response on tobacco leaves and were pathogenic on
bacterial canker in the major cherry producing areas of immature cherry fruit and green cherry tree shoots.
South Africa. Following isolations from infected trees, The current study shows that P. syringae pv. syringae
strains were characterized using biochemical as well as is responsible for bacterial canker in the Western Cape
multilocus sequence analyses (MLSA). Pathogenicity Province, South Africa.
tests were undertaken with immature cherry fruit as well
as three different cherry cultivars. Although symptoms Keywords Pseudomonas syringae . MLSA . Prunus .
of bacterial canker were present in all areas surveyed, Stone fruit
P. syringae isolates were only isolated from three sites in
the Western Cape Province. The isolates collected in this
Introduction
M. Otto : T. A. Coutinho (*)
Department of Microbiology and Plant Pathology, Centre for Cherry trees (Prunus avium) are native to the area
Microbial Ecology and Genomics, Forestry and Agricultural between the Black and Caspian seas of Asia Minor,
Biotechnology Institute, University of Pretoria, Private Bag X20, and are commercially grown in many countries
Pretoria 00282, South Africa
(Alonso 2011; Lim 2012). In South Africa, the first
e-mail: teresa.coutinho@fabi.up.ac.za
orchard, Nooitgedacht (=Gydo), was established in
Y. Petersen 1890 in the Koue Bokkeveld in the Western Cape
Agricultural Research Council, Infruitec-Nietvoorbij, Private Bag Province (Watson 2016). By 1939, 32 ha of cherry
X5026, Stellenbosch 7599, South Africa
trees had been planted and today Nooigedacht is still
J. Roux one of the major cherry producing farms in the region.
Department of Plant and Soil Sciences, Forestry and Agricultural Cherry trees are also grown commercially in three
Biotechnology Institute, University of Pretoria, Private Bag X20, other provinces, namely, Free State, Mpumalanga
Pretoria 0028, South Africa
and the North-West. In the 2012/13 and 2013/14 sea-
J. Wright sons the Western Cape Province accounted for 74% of
574 Molly Ryde Street, Garsfontein, Pretoria 0181, South Africa the cherry production in South Africa [ca. 691
428 Eur J Plant Pathol (2018) 151:427–438
Megaton (MT) and 499 MT, respectively] followed by Currently, a combination of phenotypic, molecular
the Free State with 23% and 16% in Mpumalanga (ca. and physiological methods are used for the character-
105 MT and 51 MT, respectively) (Potelwa and ization of P. syringae pathogenic to cherry (Kałużna
Ntombela 2015). and Sobiczewski 2009; Vicente et al. 2004). All
As with other agricultural crops, pests and diseases P. syringae pathovars pathogenic to cherry except for
pose serious threats to the production of cherries. One Pseudomonas syringae pv. avii (Psa) produce a fluo-
such disease is bacterial canker, which was first re- rescent pigment which is visible under UV light (King
ported to occur in South Africa on stone fruit trees, et al. 1954) and are Gram negative when tested with
including cherry trees, in 1953 (Doidge et al. 1953). 3% KOH (Suslow et al. 1982). Phenotypic testing
The disease is characterized by the formation of can- methods including the LOPAT (Levan production,
kers on stems and branches, blossom blast, systemic Oxidase test, Potato soft rot, Arginine dihydrolase,
wilting and die-back of branches or the entire tree Tobacco hypersensitive reaction) (Lelliott et al.
(Agrios 2005). The first intensive studies on the dis- 1966), carbon source utilization tests (Young and
ease in South Africa were undertaken in the 1980s Triggs 1994), L-lactate utilization and GATTa tests
(Roos and Hattingh 1986, 1987a, b). Thereafter, no enable the identification of P. syringae pathovars and
further research was undertaken and the disease was races (Latorre and Jones 1979; Lelliott and Stead
considered to be of little economic importance. Infect- 1987). The virulence of P. syringae on cherry can be
ed trees bearing poor yields were removed and assessed by inoculating these strains into susceptible
replaced. cherry tree organs such as immature fruitlets (Kałużna
In the 1980’s, the causal agents of bacterial canker and Sobiczewski 2009). Phenotypic methods are used
of cherry trees in South Africa were identified as in combination with molecular methods in a polypha-
Pseudomonas syringae pv. syringae (Pss) and Ps. sic approach for the identification of P. syringae
syringae pv. morsprunorum race 1 (Psm1) and 2 pathovars and races from cherry. A commonly used
(Psm2) (Roos and Hattingh 1986). Psm2 was consid- molecular tool to delineate phylogenetic relationships
ered to be the major pathogen causing bacterial canker within species or genera is Multi Locus Sequence
in the Free State Province while both pathovars were Analyses (MLSA). MLSA studies have been used to
well established in the Western Cape (Roos and delineate the P. syringae strains within the P. syringae
Hattingh 1986). P. syringae pv. avii (Psa) has only complex into 13 phylogroups (Berge et al. 2014;
been isolated from wild cherry in Europe (Ménard Parkinson et al. 2011) corresponding to the
et al. 2003); whilst a new bacterial species, Pseudo- genomospecies derived from DNA:DNA hybridiza-
monas cerasi causing symptoms similar to bacterial tion analyses (Gardan et al. 1999). For MLSA analy-
canker was recently identified as a pathogen of culti- ses of P. syringae pathovars and races from cherry, the
vated cherry trees (Kałużna et al. 2016a, b). Disease housekeeping genes cts, gapA, gyrB, rpoD (Kałużna
outbreaks caused by Pss, Psm1 and Psm2 have been et al. 2010a) and rpoB (Ait Tayeb et al. 2005) are often
reported globally (reviewed by Lamichhane et al. sequenced.
2014), but P. cerasi has only been isolated in Poland Over the past few years South Africa has and is
(Kałużna et al. 2016a). Pss has a broad host range and still experiencing a severe drought and unusually
is pathogenic to pome fruit as well as several cultivat- high temperatures. These abiotic factors have led to
ed stone fruits including cherry, plum, peach and increased reports of bacterial canker in stone fruit tree
apricot (Kałużna et al. 2010a; Karimi-Kurdistani and orchards, including cherry orchards. In spring 2014,
Harighi 2008; reviewed by Lamichhane et al. 2014; trees in several cherry orchards in South Africa
Wenneker et al. 2013). The other P. syringae showed typical symptoms of bacterial canker includ-
pathovars have much narrower host ranges: Psm1 ing stem and branch cankers, die-back of branches
has primarily been isolated from cherry, plum and and leaf spots. The aim of this study was to conduct a
apricot whereas Psm2 and P. cerasi have only been survey in cherry production areas of South Africa to
associated with cherry (reviewed by Bultreys and determine the prevalence of the disease and identify
Kaluzna 2010; Kałużna et al. 2016a). the bacteria associated with the disease symptoms.
Eur J Plant Pathol (2018) 151:427–438 429
These results would then be compared to those ob- suspended in sterile distilled water and the concentra-
tained in the 1980s when identification of bacteria tions were adjusted to approx. 108 CFU/ml (OD600 =
was based solely on chemotaxonomic criteria. 0.3). The sterilized immature cherry fruit were injected
with 20 μl of bacterial suspension using a hypodermic
syringe needle. Immature cherry fruit were treated with
Materials and methods sterile water to serve as a negative control. The inocu-
lated fruits were incubated for 96 h at 24 °C to assess
Surveillance and sampling pathogenicity and the necrotic lesions that formed were
measured from the needle wounds to the widest edge of
Surveys were conducted in 11 sweet cherry orchards in the necrotic lesions. The pathogenicity of the strains was
four provinces (Gauteng, Mpumalanga, Free State and rated according to the following scale modified from
Western Cape) of South Africa in October/November Kałużna and Sobiczewski (2009): non-pathogenic
2014 and 2015. In each orchard, 10% of each cultivar strains = 0–3 mm, moderately pathogenic = 3–6 mm
was randomly surveyed for the presence of bacterial and highly pathogenic= >6 mm. The pathogenicity trial
disease symptoms. Symptoms were noted as being pres- on the immature cherry fruit was repeated. Of all bacte-
ent or absent. Leaves, stem- and branch tissue showing rial isolates, only 31 bacterial isolates belonging to
typical symptoms of bacterial canker were collected. Pseudomonas gave a positive HR result on bean pods
Diseased material was cut from trees using sterile equip- (Phaseolus vularis) an were moderately pathogenic to
ment, placed into polythene bags and kept at 4 °C until immature Bing fruit (Fig. 1). Brown tissue necrosis
analyses were undertaken. developed at the site of infiltration and extended be-
tween four and six millimetre in diameter. No symptoms
Bacterial isolation and growth conditions developed in the fruit treated with sterile water (Fig. 2).
Table 1 Multilocus sequencing analyses (MLSA) primers used in this study to amplify housekeeping genes from bacterial strains isolated
from cherry trees with symptoms of bacterial canker in South Africa
Table 2 GenBank accession numbers of the DNA sequences of cherry trees in three farms (farm A, B and C) located in the
four housekeeping genes amplified from Pseudomonas strains and Western Cape Province of South Africa
used for phylogenetic analysis of bacteria isolated from diseased
bacterial strains obtained in the current study (Table 3). data set, to determine the best-fit evolutionary model to
The dataset was aligned using the MAFFT (Version 7) apply to each gene and the concatenated dataset. The
online alignment tool (Katoh and Standley 2013) and model score was evaluated with the hierarchical likeli-
trimmed at both ends using BioEdit Sequence Alignment hood ratio (hLRT) and the standard Akaike Information
Editor (Hall 1999). Partition-homogeneity tests were per- criterion (AIC). Neighbour-joining (NJ) trees were drawn
formed in PAUP 4.0b10 (Swofford 2002) to establish if for each gene and the concatenated data by using MEGA
the four genes could be combined to form a single (Version 6) and PhyML (Version 3.1) respectively. Boot-
concatenated data set. JModelTest (Posada 2008) was strap analysis with 1000 replicates was performed on all
applied to all four data sets, as well as the concatenated five trees to assess the reliability of the clusters generated.
432 Eur J Plant Pathol (2018) 151:427–438
Table 3 Pseudomonas reference strains used for multilocus se- (PAMDB) (Almeida et al. 2010) and from reference strains in
quence analysis (MLSA). All the sequences were obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/were)
the Plant Associated and Environmental Microbes database
CFBP 3846PT Pseudomonas syringae pv. avii Prunus avium (cherry) 1 NCBI
CFBP 2212PT Pseudomonas syringae pv. tomato Solanum lycopersicum (tomato) 1 PAMDB
CFBP 2351PT Pseudomonas syringae pv. morsprunorum Prunus domestica (plum) 1 NCBI
MAFF 302280 Pseudomonas syringae pv. morsprunorum Prunus domestia (plum) 1 NCBI
BPIC 631 T Pseudomonas avellanae Corylus avellana (hazel) 1 NCBI
LMG 3487 Pseudomonas syringae pv. avellanae Corylus avellana (hazel) 1 NCBI
HRI-W 7872 Pseudomonas syringae pv. syringae Prunus domestica (plum cv. Opal) 2 NCBI
LMG 1247T Pseudomonas syringae pv. syringae Syringa vulgaris (lilac) 2 PAMDB
58 T Pseudomonas cerasi Prunus avium (cherry) 2 NCBI
Cit7 Pseudomonas syringae Citrus sinensis (navel orange) 2 PAMDB
M301765 Pseudomonas syringae pv. glycinea Glycine max (soybean) 3 PAMDB
B076 Pseudomonas syringae pv. glycinea Glycine max (soybean) 3 NCBI
R4_A29–2 Pseudomonas syringae pv. glycinea Glycine max (soybean) 3 PAMDB
CFBP 5067 Pseudomonas syringae pv. nerii Nerium oleander (oleander) 3 NCBI
NCPPB 3335 Pseudomonas syringae pv. savastanoi Olea europea (olive tree) 3 NCBI
ATCC 11528 Pseudomonas syringae pv. tabaci Nicotiana tabacum (tobacco) 3 NCBI
6605 Pseudomonas syringae pv. tabaci Nicotiana tabacum (tobacco) 3 NCBI
CFBP 4389T Pseudomonas palleroniana Oryza sativa (rice) Outgroup NCBI
CFBP 2022T Pseudomonas salomonii Allium sativum (garlic) Outgroup NCBI
DSM18862 Pseudomonas azotoformans Oryza sativa (rice) Outgroup NCBI
Pathogenicity testing on P. avium shoots into each of the three upper internodes of two sep-
arate shoots on each tree. Each bacterial strain was
Pathogenicity trials on P. avium shoots were con- injected into three trees of each cultivar, resulting in
ducted as previously described by Latorre and Jones six replicates for each isolate. Control shoots were
(1979), Roos and Hattingh (1983) and Roos (1986). treated with sterilized water. All inoculated sites
The vegetative shoots on two-year-old cherry trees were covered with parafilm. Lesion lengths were
of three different cultivars (Bing, Giant Heidelfinger measured after four weeks. The identity of re-
and Stella) grown under field conditions were inoc- isolated bacterial isolates from branch cankers that
ulated with three pathogenic strains of Pseudomo- had formed was verified by oxidase, GATTa, and
nas, as established from the immature cherry fruit HR tests. The pathogenicity trial on the cherry trees
inoculations. The strains used were Pss 38, 190 and was conducted twice.
154 and were representative of three different farms
surveyed (farm A, B and C). Twenty-four hour-old Statistical analyses
cultures of the three P. syringae strains grown on
King’s B medium were suspended in sterile distilled The cherry tree inoculation data were analysed by
water and the concentration adjusted to approx. 108 one-way analysis of variance (ANOVA). Mean sep-
CFU/ml (OD600 = 0.3). After surface sterilization of arations were performed by Tukey’s test using R
the vegetative shoots of seedlings, a 1–2 mm deep software to determine if there was a significant
hole was made into the vegetative shoots of seed- difference in pathogenicity between the respective
lings by puncturing the cortex and phloem using a P. syringae strains and susceptibility of different
sharp scalpel. A hypodermic syringe was then used cherry tree cultivars. Differences at p ≤ 0.01 were
to inject 10 μl of a standardized bacterial suspension considered significant.
Eur J Plant Pathol (2018) 151:427–438 433
Chandel et al. 2011). The difficulty in isolation of Pss has certainly changed in the 35 years since the last
(isolation success of 5%) corresponds to Vicente et al. survey conducted by Roos and Hattingh (1986). Not
(2004), who had an isolation success of 18%. It is only has the major cherry production areas shifted, but
speculated that the latter could possibly have been so have the predominant bacterial species responsible
due to the time of sampling. In the current study, it for canker symptoms on cherry trees and the cultivars
was probably due to the method of isolation that was planted. Roos and Hattingh (1986, 1987a) reported the
used. Crushing diseased woody tissue with a mortar prevalence of Psm1 in the Free State Province and
and pestle in a drop of sterile water followed by occurrence of Pss, Psm1 and Psm2 in the Western Cape.
streaking on to agar as performed by Balaž et al. The current study, however, showed only the presence
(2016) would probably have yielded a higher isolation of a homogenous Pss population in the Western Cape,
rate. which is becoming the major cherry producing region of
The MLSA provided an accurate approach for phy- South Africa. In the 1980s the cultivars BCorum^ and
logenetic affiliation of the 31 Pss which grouped into BEmperor Francis^ were well established (Roos and
two clades. The geographic separation between the Hattingh 1986) whereas today neither cultivar is grown
farms where the respective isolates were obtained might and new cultivars such as BBlack Star^ occur. Further
provide a reason for the existence of two separate groups studies are needed to clarify whether all symptoms
within the Pss strains from this study. Farm A and B are observed in this study are in fact as a result of bacterial
situated next to each other in an area which experiences canker, or if other causal agents are of importance. This
lower temperatures than farm C which is 22 km further is critical for implementing effective management and
away. Even though there are two separate groups within the fine-tuning management strategies to ensure higher
the Pss isolates, the analyses showed that the strains are production from cherry farming.
highly similar. This is interesting as it is known that Pss
is heterogenous (Abbasi et al. 2013; Kałużna et al.
2010a; Khayamie et al. 2009; Iličić et al. 2016), Acknowledgements The Horticultural Knowledge Group
(HORTGRO) and National Research Foundation (NRF) are ac-
attacking various hosts and is dispersed in several ways knowledged for funding this research. In addition, the cherry
including aerosols, water and aphids (Crosse 1966; farmers are acknowledged for access to their farms and informa-
Morris et al. 2008; Stavrinides et al. 2009). Similar tion provided.
results of homo-genetic isolates have, however, recently
been reported for Pss (Balaž et al. 2016), Psm1 and Compliance with ethical standards
P. cerasi (Kałużna et al. 2016b). The high level of
Conflict of interest The authors declare no conflict of interest.
identity of the strains shows that the Pss isolates evolved
in a specific association with the cherry trees or could Human participants and animal studies No humans or ani-
possibly be due to pathogen spread. It could, however, mals were involved in the execution of this research. All authors
also point out a common source of inoculum. The latter have consented to the submission of this manuscript to EJPP.
is probably the case for the South African Pss strains as
the cherry trees from which they were isolated were
obtained roughly at the same time from the same nurs-
ery. The spread of bacterial canker though nurseries has
References
been reported by several authors (Bultreys and Kaluzna
2010; Luz 1997; Vicente et al. 2004). Insight into the
populations structure of the current Pss strains provides Abbasi, V., Rahimian, H., & Tajick-Ghanbari, M. A. (2013).
Genetic variability of Iranian strains of Pseudomonas
valuable information on disease epidemiology and can syringae pv. syringae causing bacterial canker disease of
also be used for breeding and resistance to the pathogen, stone fruits. European Journal of Plant Pathology, 135,
which is currently considered to be the most effective 225–235.
and economically practical disease management strate- Agrios, G. N. (2005). Plant Pathology (5th ed.). Amsterdam:
Elsevier Academic Press.
gy (Bassi 1999).
Ait Tayeb, L., Ageron, E., Grimont, F., & Grimont, P. A. (2005).
This study shows the current status with respect to Molecular phylogeny of the genus Pseudomonas based on
the prevalence of bacterial canker and the causal agent, rpoB sequences and application for the identification of
Pss, in cherry orchards in South Africa. The situation isolates. Research in Microbiology, 156, 763–773.
Eur J Plant Pathol (2018) 151:427–438 437
Almeida, N. F., Yan, S., Cai, R., Clarke, C. R., Morris, C. E., Iličić, R., Balaž, J., Stojšin, V., & Jošić, D. (2016).
Schaad, N. W., Schuenzel, E. L., Lacy, G. H., Sun, X., Jones, Characterization of Pseudomonas syringae pathovars from
J. B., Castillo, J. A., Bull, C. T., Leman, S., Guttman, D. S., different sweet cherry cultivars by RAPD analyses. Genetika,
Setubal, J. C., & Vinatzer, B. A. (2010). PAMDB, a 48(1), 285–295.
multilocus sequence typing and analysis database and Kałużna, M., & Sobiczewski, P. (2009). Virulence of
website for plant-associated microbes. Phytopathology, 100, Pseudomonas syringae pv. syringae pathovars and races
208–215. originating from stone fruit trees. Phytopathologia, 54, 71–
Alonso, J. S. (2011). Producción, comercialización, Mercado y 79.
oportunidades de la cereza. [Sweet cherry production, mar- Kałużna, M., Ferrante, P., Sobiczewski, P., & Scortichini, M.
keting, and market opportunities]. VidaRURAL, 23, 46–50. (2010a). Characterization and genetic diversity of
Annesi, T., Motta, E., & Forti, E. (1997). First report of Pseudomonas syringae from stone fruits and hazelnut using
Blumeriella jaapii teleomorph on wild cherry in Italy. Plant repetitive-PCR and MLST. Journal of Plant Pathology, 92,
Disease, 81, 1214. 781–787.
Balaž, J., Iličić, R., Ognjanov, V., Ivanović, Ž., & Popović, T. Kałużna, M., Pulawska, J., & Sobiczewski, P. (2010b). The use of
(2016). Etiology of bacterial canker on young sweet cherry PCR melting profile for typing Pseudomonas syringae iso-
trees in Serbia. Journal of Plant Pathology, 98(2), 285–294. lates from stone fruit trees. European Journal of Plant
Barakat, R. M., & Johnson, D. A. (1997). Expansion of cankers Pathology, 126, 437–443.
caused by Leucostoma cincta on sweet cherry trees. Plant Kałużna, M., Willems, A., Pothier, J. l. F., Ruinelli, M.,
Disease, 81, 1391–1394. Sobiczewski, P., & Puławska, J. (2016a). Pseudomonas
Bassi, D. (1999). Apricot culture: present and future. Acta cerasi sp. nov. (non Griffin, 1911) isolated from diseased
Horticulturae, 488, 35–40. tissue of cherry. Systemic and Applied Microbiology, 39,
Berge, O., Monteil, C. L., Bartoli, C., Chandeysson, C., Guilbaud, 370–377.
C., Sands, D. C., & Morris, C. E. (2014). A user's guide to a Kałużna, M., Willems, A., Pothier, J. F., Ruinelli, M.,
data base of the diversity of Pseudomonas syringae and its Sobiczewski, P., & Puławska, J. (2016b). Characterization
application to classifying strains in this phylogenetic com- and genetic diversity of causal agent of stone fruit bacterial
plex. PLoS One, 9, e105547. canker Pseudomonas cerasi, a new pathogen of cherry. Acta
Bultreys, A., & Kaluzna, M. (2010). Bacterial cankers caused by Horticulturae, 1149, 9–14.
Pseudomonas syringae on stone fruit species with special Karimi-Kurdistani, G., & Harighi, B. (2008). Phenotypic and
emphasis on the pathovars syringae and morsprunorum race molecular properties of Pseudomonas syringae pv. syringae
1 and race 2. Journal of Plant Pathology, 92, S1–S21. the causal agent of bacterial canker of stone fruit trees in
Casals, C., Segarra, J., De Cal, A., Lamarca, N., & Usall, J. (2015). Kurdistan province. Journal of Plant Pathology, 90, 81–86.
Overwintering of Monilinia spp. on mummified stone fruit. Katoh, K., & Standley, D. M. (2013). MAFFT multiple sequence
Journal of Phytopathology, 163, 160–167. alignment software version 7: Improvements in performance
Chandel, V., Rana, T., Hallan, V., & Zaidi, A. A. (2011). Detection and usability. Molecular Biology and Evolution, 30, 772–
of Prunus necrotic ring spot virus in plum, cherry and almond 780.
by serological and molecular techniques from India. Archives Khayamie, S., Niknejad, K. N., Rabie, S., & Ebadie, A. A. (2009).
of Phytopathology and Plant Protection, 44, 1779–1784. Genetic characterization of P. syringae pv. syringae strains
Crosse, J. E. (1966). Epidemiological relations of the pseudomo- from stone fruits based on RAPD analysis in Iran.
nad pathogens of deciduous fruit trees. Annual Review of Agricultura Tropica et Subtropica, 42(4), 162–166.
Phytopathology, 14, 291–310. King, E. O., Ward, M. K., & Raney, D. E. (1954). Two simple
Doidge, E.M., Bottomley, A.M., van der Planck, J.E., and Pauer, media for the demonstration of pyocyanin and fluorescin.
G.D. 1953. A revised list of plant diseases in South Africa. Journal of Laboratory and Clinical Medicine, 44(2), 301–
Union of South Africa, Department of Agriculture, Science 307.
Bulletin No. 346, 1–122. Lamichhane, J. R., Varvaro, L., Parisi, L., Audergon, J.-M., &
Gardan, L., Shafik, H., Belouin, S., Broch, R., Grimont, F., & Morris, C. E. (2014). Disease and frost damage of woody
Grimont, P. A. (1999). DNA relatedness among the pathovars plants caused by Pseudomonas syringae: Seeing the forest
of Pseudomonas syringae and description of Pseudomonas for the trees. Advances in Agronomy, 126, 235–295.
tremae sp. nov. and Pseudomonas cannabina sp. nov. (ex Latorre, B. A., & Jones, A. L. (1979). Pseudomonas
Sutic and Dowson 1959). International Journal of Systematic morsprunorum,the cause of bacterial canker of sour cherry
Bacteriology, 49, 469–478. in Michigan, and its epiphytic association with P. syringae.
Goszczynska, T., Serfontein, J. J., & Serfontein, S. (2000). Phytopathology, 69, 335–339.
Introduction to practical phytobacteriology: A manual for Lelliott, R. A., & Stead, D. E. (1987). Methods for the diagnosis of
phytobacteriology (2nd ed.). Pretoria, South Africa: Safrinet. bacterial diseases of plants. In T. F. Preece (Ed.), Methods in
Hall, T. A. (1999). BioEdit: A user-friendly biological sequence plant pathology (pp. 37–131). Oxford: Blackwell Scientific
alignment and editor and analyses program for windows 95/ Publications.
98/NT. Nucleic Acids Symposium Series, 41, 95–98. Lelliott, R. A., Billing, E., & Hayward, A. C. (1966). A determi-
Hwang, M. S. H., Morgan, R. L., Sakar, S. F., Wang, P. W., & native scheme for the fluorescent plant pathogenic pseudo-
Guttman, D. S. (2005). Phylogenetic characterization of vir- monads. Journal of Applied Bacteriology, 29, 470–489.
ulence and resistance phenotypes of Pseudomonas syringae. Lim, T. K. 2012. Edible medicinal and non-medicinal plants.
Applied and Environmental Microbiology, 71, 5182–5191. Volume 4, Fruits. Springer, Dordrecht.
438 Eur J Plant Pathol (2018) 151:427–438
Luz, J.P.M. 1997. Detection and epidemiology of bacterial canker Sarkar, S. F., & Guttman, D. S. (2004). Evolution of the core
(Pseudomonas syringae) on wild cherry (Prunus avium). genome of Pseudomonas syringae, a highly clonal, endemic
PhD thesis. University of Reaging. plant pathogen. Applied and Environmental Microbiology,
Ménard, M., Sutra, L., Luisetti, J., Prunier, J. P., & Gardan, L. 70, 1999–2012.
(2003). Pseudomonas syringae pv. avii (pv. nov.), the causal Sholberg, P. L., & Quamme, H. A. (1999). Dieback of
agent of bacterial canker of wild cherries (Prunus avium) in pome fruit rootstocks caused by Pseudomonas
France. European Journal of Plant Pathology, 109, 565–576. syringae. Canadian Journal of Plant Science, 79,
Morris, C. E., Sands, D. C., Vinatzer, B. A., Glaux, C., Guilbaud, 387–394.
C., Buffière, A., Yan, S., Dominguez, H., & Thompson, B. Stavrinides, J., McCloskey, J. K., & Ochman, H. (2009). Pea aphid
M. (2008). The life history of the plant pathogen as both host and vector for the phytopathogenic bacterium
Pseudomonas syringae is linked to the water cycle. Pseudomonas syringae. Applied and Environmental
Multidisciplinary Journal of Microbial Ecology, 2, 321–334. Microbiology, 75, 2230–2235.
Nowell, R. W., Laue, B. E., Sharp, P. M., & Green, S. (2016). Suslow, T. V., Schroth, M. N., & Isaka, M. (1982). Appication of a
Comparative genomics reveals genes significantly associated rapid method for gram differentiation of plant pathogenic and
with woody hosts in the plant pathogen Pseudomonas saprophytic bacteria without staining. Phytopathology, 72,
syringae. Molecular Plant Pathology, 17, 1409–1424. 917–918.
Parkinson, N., Bryant, R., Bew, J., & Elphinstone, J. (2011). Rapid Swofford, D. L. 2002. Phylogenetic Analyses Using Parsimony
phylogenetic identification of members of the Pseudomonas (and other methods). Version 4.0b10. Sinauer Associates,
syringae species complex using the rpoD locus. Plant Sunderland.
Pathology, 60, 338–344.
Vicente, J. G., Alves, J. P., Russell, K., & Roberts, S. J. (2004).
Posada, D. (2008). jModelTest: phylogenetic model averaging.
Identification and discrimination of Pseudomonas syringae
Molecular Biology and Evolution, 25, 1253–1256.
isolates from wild cherry in England. European Journal of
Potelwa, Y., and Ntombela, S. 2015. South African Fruit Trade
Plant Pathology, 110, 337–351.
Flow. Issue 17. Online publication. http://www.namc.co.
za/upload/South-African-Fruit-Trade-Flow-February-2015- Watson, N. 2016. South Africa: extreme drought and heat has left
Issue-17.pdf. its mark on cherry volumes. http://www.freshplaza.
Roos, I. M.M. 1986. Bacterial canker of stone fruit trees caused by com/article/166877/South-Africa-Extreme-drought-and-
Pseudomonas syringae pv. syringae and Pseudomonas heat-has-left-its-mark-on-cherry-volumes. Accessed 5
syringae pv. morsprunorum: Numerical analyses of pheno- June 2017.
typic features of the pathogens and systemic invasion of host Wenneker, M., Meijer, H., Maas, F. M., de Bruine, A.,
tissue. PhD thesis. University of Stellenbosch. Vink, P., & Pham, K. (2013). Bacterial canker of plum
Roos, I. M. M., & Hattingh, M. J. (1983). Fluorescent pseudomo- trees (Prunus domestica), caused by Pseudomonas
nads associated with bacterial canker of stone fruit in South syrin gae pathovars, in the Netherlan ds. Acta
Africa. Plant Disease, 67, 1267–1269. Horticulturae, 985, 235–239.
Roos, I. M. M., & Hattingh, M. J. (1986). Bacterial canker of Yan, S., Liu, H., Mohr, T. J., Jenrette, J., Chiodini, R., Zaccardelli,
sweet cherry in South Africa. Phytophylactica, 18, 1–4. M., Setubal, J. C., & Vinatzer, B. A. (2008). Role of recom-
Roos, I. M. M., & Hattingh, M. J. (1987a). Pathogenicity and bination in the evolution of the model plant pathogen
numerical analyses of phenotypic features of Pseudomonas Pseudomonas syringae pv. tomato DC3000, a very atypical
syringae strains isolated from deciduous fruit trees. tomato strain. Applied and Environmental Microbiology, 74,
Phytopathology, 77, 900–908. 3171–3181.
Roos, I. M. M., & Hattingh, M. J. (1987b). Systemic invasion of Young, J. M., & Triggs, C. M. (1994). Evaluation of determinative
cherry leaves and petioles by Pseudomonas syringae pv. tests for pathovars of Pseudomonas syringae van Hall 1902.
morsprunorum. Phytopathology, 77, 1246–1252. Journal of Applied Bacteriology, 77, 195–207.