Research Institute of Pomology and Floriculture, Skierniewice, Poland

VIRULENCE OF PSEUDOMONAS SYRINGAE PATHOVARS

AND RACES ORIGINATING FROM STONE FRUIT TREES1

M. Kałużna and P. Sobiczewski

Abstract

Virulence of 23 Pseudomonas syringae strains representing pathovars syringae and

morsprunorum races 1 and 2 was studied on sweet cherry fruitlets cv. ‘Napoleon’. All
P. syringae pv. syringae (Pss) strains caused deep black brown necroses while those
of P. syringae pv. morsprunorum (Psm) – brownish water soaked superficial lesions.
The size of Pss necroses indicating bacteria virulence corresponded with syringo-
mycin production (largest inhibition zone of Rhodotorula pilimanae growth). Strains
of Psm race 2 appeared to be more virulent than those of race 1.

Key words: bacterial canker, stone fruits, Pseudomonas syringae, pathovars, races,
virulence, cherry fruitlet test, syringomycin

Introduction

Pseudomonas syringae is a pathogen of over 180 plant species including fruit trees
(Agrios 2005). Because of differential pathogenic abilities, strains of P. syringae are
classified into over 50 pathovars differing with range of hosts, i.e. species or
cultivars (Young et al. 1996). Pathovars: syringae (causing, among others, bacterial
canker particularly on sour cherries and belonging to genomospecies 1) and
morsprunorum (found most often on sweet cherries and belonging to genomo-
species 2 and 3) are the main causal agents of bacterial canker and gummosis on
stone fruit trees (Burkowicz 1981, Sobiczewski 1984, Young 1991, Gardan et al.
1999, Vicente et al. 2004). Moreover, from some species of stone fruits pathovars
avii and persicae were isolated (Young 1988, Ménard et al. 2003, EPPO... 2005).

This work was performed within the framework of COST Action 873 “Bacterial diseases of stone
1

fruits and nuts” and supported by Polish Ministry of Science and Higher Education grant No
118/N-COST/2008/0.

Phytopathologia 54: 71–79

© The Polish Phytopathological Society, Poznań 2009
ISSN 2081-1756
72 M. Kałużna and P. Sobiczewski

However, till now they were not detected in Poland. The diagnosis of bacterial can-
ker is commonly based on phenotypic characterization of its causal agent, includ-
ing pathogenicity (Bultreys and Gheysen 1999, Schaad et al. 2001, Vicente et al.
2004). The pathogenicity tests are being conducted on different host plants and or-
gans (Endert and Ritchie 1984, Sobiczewski 1984, Burkowicz and Rudolph 1994,
Liang et al. 1994, Ménard et al. 2003, Scortichini et al. 2003, Vicente et al. 2004,
Renick et al. 2008).
The aim of our study was to determine virulence (sensu degree of pathogenicity)
of P. syringae strains representing two pathovars and races isolated from stone fruit
trees growing in various regions of Poland.

Material and methods

Bacterial strains

Twenty three strains of P. syringae originating from symptomatic tissues of vari-

ous organs of sweet and sour cherry, apricot and plum were studied (Table 1). The
reference strains Pss 2905 (Pss) from the authors’ collection, LMG 1247 (Pss),
LMG 2222 (Psm race 1) and CFBP 3800 (Psm race 2) were included for comparison.

Syringomycin production

The ability of syringomycin production by studied strains was evaluated on

peptone-glucose-NaCl agar medium (PGNaClA; Bultreys and Gheysen 1999). Bac-
teria of tested strains were streaked on the medium in the center of each Petri dish
(line of 3-cm-long and 0.8-cm-wide) and incubated at 26°C for 48 h. After that
time water suspension of the yeast Rhodotorula pilimanae MUCL30397 was sprayed
onto the medium surface. The inhibition zones of R. pilimanae growth around bac-
teria growth line were measured after next 48 h of incubation at 25°C.

Preparation of bacterial water suspension

Bacteria of each strain were cultivated on King’s B medium (Difco, Pseudomo-

nas Agar F) at 26°C. After 48 h bacteria were washed out of the medium with ster-
ile distilled water. Their concentration in suspension was adjusted to 108 cfu/ml
with spectrophotometer Semco S91E at 660 nm and using serial dilution and plat-
ing method on King’s B.

Virulence testing on sweet cherry fruitlets

Freshly collected immature sweet cherry fruits cv. ‘Napoleon’ were disinfected
by dipping in 50% ethanol for 3 min and then rinsed three times in sterile distilled
water. Afterwards, fruitlets were wrapped with paper towel to remove the excess
of water. Each fruitlet was inoculated by pricking in two places to the depth of 2
 Virulence of Pseudomonas syringae pathovars and races... 73

Table 1
Characteristics of 23 Pseudomonas syringae strains examined in this study
(Kałużna et al. 2009)
Genes encoding toxin production
a Year of
Group Strain Host syrB cfl irp1
isolation
(syringomycin) (coronatine) (yersiniabactin)
Psm race 1 701A Sweet cherry 2005 – + –
702 Plum 1994 – – –
704 Sweet cherry 1994 – + –
710 Sweet cherry 1996 – + –
755 Plum 1999 – – –
771 Plum 1999 – – –
782 Sweet cherry 2001 – – –
787 Plum 2001 – – –
788 Plum 2001 – – –
793 Plum 2001 – – –
LMG 2222* Prunus avium – + –
cv. ‘Napoleon’

Psm race 2 701 Sour cherry 1994 – – +

719 Sour cherry 1997 – – +
732 Sour cherry 1997 – – +
733 Sour cherry 1997 – – +
745 Sour cherry 1999 – – +
764 Sour cherry 1999 – – +
CFBP 3800* Prunus cerasus – – +

Atypicalb 791 Sour cherry 2001 – – –

Pss 702A Plum 2005 + – –

753 Apricot 1999 + – –
757 Plum 1999 + – –
760 Sour cherry 1999 + – –
762 Apricot 1999 + – –
763 Sour cherry 1999 + – –
2905*- Sour cherry 1978 + – –
LMG 1247* Syringa vulgaris + – –
Based on LOPAT, GATTa and L-lactate tests (Lelliott and Stead 1987, Lattore and Jones 1979).
a

Determined as Pss on base of PCR MP.

b

“+” – positive, “–” – negative.

*Reference strain.

mm with sterile needle previously immersed in suspension of each strain. After in-
oculation each fruitlet was immediately placed on moist filter paper in sterile Petri
dish and incubated at 22°C for four days. The reference strains and sterile distilled
74 M. Kałużna and P. Sobiczewski

water were included as positive and negative control, respectively. Ten fruitlets
were used for testing of each strain.
After 24, 48 and 96 h of incubation the symptoms developed around inocula-
tion wounds were observed. Their severity was evaluated separately for each
wound, using scales depending on the type of symptoms. For deep black brown
necroses the following scale was used: 0 – no symptoms, 1 – necroses up to 1 mm
in diameter, 2 – necroses up to 2 mm, 3 – up to 3 mm, 4 – up to 4 mm, 5 – up to 5
mm, 6 – necroses over 5 mm. Similar degree scale was used for scoring the second
type of symptoms, i.e. brownish water soaked superficial lesions. Results were
subjected to ANOVA analysis of variance. For separation of means the New-
man-Keuls test at 5% significance was used.

Results and discussion

All strains caused symptoms showing disorders around wounds on fruitlets.

After 24 h from fruit inoculations with strains of Pss (760, 702A, 762, 753, 757,
2905, 763) deep black brown necroses were observed (Phot. 1 B, Table 2) while
strains of Psm race 1 (782, 788, 710, 701A) and Psm race 2 (764, 701, 719, 745)
gave brownish water soaked superficial lesions (Phot. 1 A, Table 3). At that time
strains 791, 787, 755, 733, 732, 771, 704, 702, 793 did not induce any symptoms.
48 h after inoculation strain 791 produced deep black brown necroses (Table 2)
but strains 787, 755, 733, 732, 771, 704 – brownish superficial lesions (Table 3).
The severity of symptoms increased throughout the experiment. Differential viru-
lence of tested strains, determined on the basis of inoculated tissue destruction
size, was observed. Of all studied bacteria the strains 760, 762 and 702A (Pss)
showed the highest virulence (Table 2), whereas the lowest: 793, 771 and 701A

Phot. 1. Symptoms occurring on immature sweet cherry fruits cv. ‘Napoleon’ after inoculation with
Pseudomonas syringae strains: A – fruits inoculated with strains of Psm (races 1 and 2; brownish water
soaked superficial lesions), B – fruits inoculated with strains of Pss (black-brown sunken necroses)
(photo by M. Kałużna)
 Virulence of Pseudomonas syringae pathovars and races... 75

Table 2
Virulence of Pseudomonas syringae pv. syringae on sweet cherry fruitlets cv.
‘Napoleon’ (degree of disease severity in 0–6 scale)
Hours from inoculation: Inhibition zone
Strain of Rhodotorula pilimanae
24 48 96 (mm)
702A 1.9 c 3.0 e 4.5 cd 15
753 2.3 c 2.7 e 4.1 cd 7
757 2.3 c 2.3 d 3.7 c 8
760 2.4 c 3.0 e 5.1 d 16
762 2.0 c 2.8 e 5.1 d 15
763 2.1 c 2.1 d 4.1 cd 9
791 0.0 a 0.8 b 2.8 b 0
2905 2.0 c 2.3 d 3.9 cd 11
LMG 1247 1.0 b 1.6 c 4.4 cd 14

H2O 0.0 a 0.0 a 0.0 a Not tested

In columns means followed by the same letters are not significantly different at 5 percent level.

(Psm race 1; Table 3). No symptoms were developed around wounds made with
needle dipped in sterile water.
It should be pointed out that some of Pss strains appeared more virulent than
the reference strains Pss 2905 and LMG 1247. All Pss strains, except for 791, pro-
duced syringomycin (SR+) what was indicated by inhibition of yeast Rhodotorula
pilimanae MUCL30397 growth on PGNaClA medium (Phot. 2). This ability corre-
sponded with the strain’s virulence. The most virulent strains (760, 762, 702A)
caused inhibition zones from 14 to 16 mm while the less virulent strains gave inhi-
bition zones of 0–11 mm (Table 2). Although the gene encoding syringomycin
(syrB) was detected in those strains (Kałużna et al. 2009), its expression could dif-
fer between them. All SR+ strains were more virulent than strain 791 lacking this

Phot. 2. Inhibition zone of Rhodotorula pilimanae MUCL30397 on PGNaClA medium

caused by strain 760 (Pss) and lack of such zone in case of 704 (Psm)
(photo by M. Kałużna)
76 M. Kałużna and P. Sobiczewski

Table 3
Virulence of Pseudomonas syringae pv. morsprunorum on sweet cherry fruitlets
cv. ‘Napoleon’ (degree of disease severity in 0–6 scale)
Hours from inoculation: Inhibition zone
Strain of Rhodotorula pilimanae
24 48 96 (mm)
701A 0.2 a 0.7 b-e 1.6 cd 0
702 0.0 a 0.0 a 2.2 de 0
704 0.0 a 0.2 ab 2.2 de 0
710 0.1 a 0.9 c-f 2.2 de 0
755 0.0 a 1.1 d-f 3.8 gh 0
771 0.0 a 0.3 ab 1.3 bc 0
782 0.2 a 1.4 fg 2.8 ef 0
787 0.0 a 1.1 d-f 3.0 ef 0
788 0.3 a 1.3 e-g 3.0 ef 0
793 0.0 a 0.0 a 0.2 a 0
LMG 2222 0.0 a 0.4 a-c 0.8 b 0

701 0.3 a 1.45 fg 2.7 ef 0

719 0.2 a 1.1 d-f 2.9 ef 0
732 0.0 a 0.45 a-c 2.5 ef 0
733 0.0 a 0.5 a-d 3.3 fg 0
745 0.1 a 0.7 b-e 3.3 fg 0
764 0.2 a 1.8 g 4.2 h 0
CFBP 3800 0.2 a 1.3 e-g 2.3 de 0

H2O 0.0 a 0.0 a 0.0 a Not tested

In columns means followed by the same letters are not significantly different at 5 percent level.

ability. On the other hand, study of Xu and Gross (1988) with Pss Tox– transpozon
mutants showed that this toxin is not required for pathogenicity but significantly
influences virulence.
None of our Psm races 1 and 2 strains inhibited R. pilimanae growth. It was found
that Psm2 strains appeared to be more virulent than strains of Psm1. As indicated
by the one-sided t-test, strains of race 2 caused significantly larger superficial le-
sions (3.02) than those of race 1 (2.25). The presence of coronatine gene (cfl) in-
volved in chlorosis induction determined in those strains (Kałużna et al. 2009)
does not correspond to their virulence, i.e. lesions size on fruitlets 96 h after
inoculation, e.g. strain 704 (cfl+) – 2.2 versus 755 (cfl–) – 3.8 mm. However, other
authors found that some coronatine mutants (cfl–) showed significantly lower viru-
lence than strains possessing this toxin (Mitchell 1982, Xu and Gross 1988,
Ullrich et al. 1993, Bender et al. 1999). Thus, it is possible that Psm virulence can
be also related with other factor/s.
 Virulence of Pseudomonas syringae pathovars and races... 77

Our findings correspond with that of Liang et al. (1994) who demonstrated in
cherry plantlet assays higher virulence of Pss strains than that of Psm (both
pathovars were earlier classified as separate species Ps and Pm) isolated from Pru-
nus. It was also proved on sweet cherry fruitlets, however, on shoots of sweet
cherry the strains characterized as morsprunorum produced longer lesions than
those caused by syringae (Burkowicz and Rudolph 1994). Strains from Pss group
showed high variability in pathogenicity tests on micropropagated plantlets of lilac
and wild cherry, as well as rooted plants of lilac, and those of Psm were pathogenic
only to cherry plantlets (Vicente et al. 2004). Comparison of our strains genetic di-
versity using PCR MP (Melting Profile PCR) indicated strong relation to patho-
genic groups. Pss strains causing black brown necroses were placed in one group
while Psm causing brownish water soaked superficial lesions – in a separate group
(Kałużna et al. 2009).
Our studies showed that pathogencity test on sweet cherry fruitlets can be eas-
ily used not only for determination of bacterial canker causal agent virulence but
also for differentiation of its pathovars. Differences in type of symptoms caused by
syringae and morsprunorum were also reported by Crosse and Garrett (1963) and
Burkowicz and Rudolph (1994). However, the possibility to determine bacteria
virulence was not studied before.

Streszczenie

WIRULENCJA PATOWARÓW I RAS PSEUDOMONAS SYRINGAE

POCHODZĄCYCH Z DRZEW PESTKOWYCH

Celem pracy było określenie wirulencji (stopnia patogeniczności) 23 szczepów

Pseudomonas syringae wyizolowanych z drzew pestkowych, reprezentujących pato-
wary syringae i morsprunorum ras 1 i 2. Na inokulowanych zawiązkach owoców cze-
reśni odmiany ‘Napoleon’ wszystkie szczepy P. syringae pv. syringae (Pss) powodo-
wały brązowo-czarne zagłębione nekrozy, a szczepy P. syringae pv. morsprunorum
(Psm) – brązowawe uwodnione powierzchniowe plamy. Wielkość nekroz Pss, jako
wskaźnik wirulencji, korespondowała z szerokością strefy zahamowania wzrostu
drożdży Rhodotorula pilimanae MUCL30397 (wskaźnik produkcji syryngomycyny).
Szczepy rasy 2 Psm były bardziej wirulentne niż szczepy rasy 1 tego patowaru.

Literature

Agrios G.N., 2005: Plant diseases caused by prokaryotes: Bacteria and Mollicutes. In: G.N. Agrios: Plant
pathology. Elsevier, Amsterdam: 667–674.
Bender C.L., Alarcón-Chaidez F., Gross D.C., 1999: Pseudomonas syringae phytotoxins: mode of action,
regulation, and biosynthesis by peptide and polyketide synthetases. Microbiol. Mol. Biol. Rev. 63:
266–292.
78 M. Kałużna and P. Sobiczewski

Bultreys A., Gheysen I., 1999: Biological and molecular detection of toxic lipodepsipeptide-producing
Pseudomonas syringae strains and PCR identification in plants. Appl. Environ. Microbiol. 65:
1904–1909.
Burkowicz A., 1981: Population dynamics of epiphytic Pseudomonas morsprunorum on the leaf surfaces of
two sweet cherry cultivars. Fruit Sci. Rep. (Skierniewice) 8: 37–48.
Burkowicz A., Rudolph K., 1994: Evaluation of pathogenicity and of cultural and biochemical tests for
identification of Pseudomonas syringae pathovars syringae, morsprunorum and persicae from fruit trees.
J. Phytopathol. 141: 59–76.
Crosse J.E., Garrett C.M.E., 1963: Studies on the bacteriophagy of Pseudomonas morsprunorum, Ps. syringae
and related organisms. J. Appl. Bacteriol. 26, 2: 159–177.
Endert E., Ritchie D.F., 1984: Detection of pathogenicity, measurement of virulence, and determina-
tion of strain variation in Pseudomonas syringae pv. syringae. Plant Dis. 68: 677–680.
EPPO Standards PM 7/43(1) Pseudomonas syringae pv. persicae. 2005. Bull. OEPP / EPPO Bull. 35:
271–273.
Gardan L., Shafik H., Belouin S., Broch R., Grimont F., Grimont P.A.D., 1999: DNA relatedness among
the pathovars of Pseudomonas syringae and description of Pseudomonas tremae sp. nov. and Pseudomo-
nas cannabina sp. nov. (ex Sutic and Dowson 1959). Int. J. Syst. Bacteriol. 49: 469–478.
Kałużna M., Puławska J., Sobiczewski P., 2009: The use of PCR melting profile for typing of Pseudomonas
syringae isolates from stone fruit trees. Eur. J. Plant Pathol. [DOI: 10.1007/s10658-009-9553-9].
Lattore B.A., Jones A.L., 1979: Pseudomonas morsprunorum, the cause of bacterial canker of sour cherry in
Michigan and its epiphytic association with P. syringae. Phytopathology 69: 335–339.
Lelliott R.A., Stead D.E., 1987: Methods for the diagnosis of bacterial diseases of plants. Meth. Plant
Pathol. 2.
Liang L.Z., Sobiczewski P., Paterson J.M., Jones A.L., 1994: Variation in virulence, plasmid content, and
genes for coronatine synthesis between Pseudomonas syringae pv. morsprunorum and P. s. syringae
from Prunus. Plant Dis. 78: 389–392.
Ménard M., Sutra L., Luisetti J., Prunier J.P., Gardan L., 2003: Pseudomonas syringae pv. avii (pv. nov.), the
causal agent of bacterial canker of wild cherries (Prunus avium) in France. Eur. J. Plant Pathol. 109:
565–576.
Mitchell R.E., 1982: Coronatine production by some phytopathogenic Pseudomonads. Physiol. Plant
Pathol. 20: 83–89.
Renick L.J., Cogal A.G., Sundin G.W., 2008: Phenotypic and genetic analysis of epiphytic Pseudomonas
syringae populations from sweet cherry in Michigan. Plant Dis. 92: 372–378.
Schaad N.W., Jones J.B., Chun W., 2001: Laboratory guide for identification of plant pathogenic bacte-
ria. APS Press, St. Paul, MN.
Scortichini M., Marchesi U., Dettori M.T., Rossi M.P., 2003: Genetic diversity, presence of the syrB
gene, host preference and virulence of Pseudomonas syringae pv. syringae strains from woody and
herbaceous host plants. Plant Pathol. 52: 277–286.
Sobiczewski P., 1984: Etiology of sour cherry bacterial canker in Poland. Fruit Sci. Rep. (Skierniewice)
11: 169–180.
Ullrich M., Bereswill S., Volksch B., Fritsche W., Geider K., 1993: Molecular characterization of field
isolates of Pseudomonas syringae pv. glycinea differing in coronatine production. J. Gen. Microbiol.
139: 1927–1937.
Vicente J.G., Roberts S.J., Russell K., Alves J.P., 2004: Identification and discrimination of Pseudomonas
syringae isolates from wild cherry in England. Eur. J. Plant Pathol. 110: 337–351.
Xu G.-W., Gross D., 1988: Evaluation of the role of syringomycin in plant pathogenesis by using Tn5
mutants of Pseudomonas syringae pv. syringae defective in syringomycin production. Appl. Environ.
Microbiol. 54, 6: 1345–1353.
Young J.M., 1988: Pseudomonas syringae pv. persicae from nectarine, peach, and Japanese plum in New
Zealand. Bull. OEPP / EPPO Bull. 18: 141–151.
Young J.M., 1991: Pathogenicity and identification of the lilac pathogen, Pseudomonas syringae pv.
syringae van Hall 1902. Ann. Appl. Biol. 118: 283–298.
Young J.M., Saddler G.S., Takikawa Y., DeBoer S.H., Vauterin L., Gardan L., Gvozdyak R.I., Stead D.E.,
1996: Names of plant pathogenic bacteria 1864–1995. Rev. Plant Pathol. 75: 721–763.
 Virulence of Pseudomonas syringae pathovars and races... 79

Authors’ address:
Monika Kałużna M.Sc., Prof. Dr. hab. Piotr Sobiczewski, Research Institute
of Pomology and Floriculture, ul. Pomologiczna 18, 96-100 Skierniewice,
Poland, e-mail: monika.kaluzna@insad.pl

Accepted for publication: 12.11.2009