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Citation: Jeluši´c, A.; Scortichini, M.;
Markovi´c, S.; Mitrovi´c, P.; Iliˇci´c, R.;
Stankovi´c, S.; Popovi´c Milovanovi´c, T.
Phylogeographic Analysis of
Soft-Rot-Causing Pectobacterium spp.
Strains Obtained from Cabbage in
Serbia. Microorganisms 2023,11, 2122.
https://doi.org/10.3390/
microorganisms11082122
Academic Editor: Assunta Bertaccini
Received: 25 July 2023
Revised: 14 August 2023
Accepted: 17 August 2023
Published: 21 August 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
microorganisms
Article
Phylogeographic Analysis of Soft-Rot-Causing Pectobacterium
spp. Strains Obtained from Cabbage in Serbia
Aleksandra Jeluši´c 1, Marco Scortichini 2, Sanja Markovi´c 1, Petar Mitrovi´c 3, Renata Iliˇci´c 4,
Slaviša Stankovi´c 5and Tatjana Popovi´c Milovanovi´c 6,*
1Institute for Multidisciplinary Research, University of Belgrade, Kneza Višeslava 1, 11030 Belgrade, Serbia;
jelusic.aleksandra@gmail.com (A.J.); sanja.markovic@imsi.rs (S.M.)
2
Council for Agronomical Research and Economics (CREA), Research Centre for Olive, Fruit and Citrus Crops,
Via di Fioranello, 52, I-00134 Roma, Italy; marco.scortichini@crea.gov.it
3Institute for Field and Vegetable Crops—National Institute of the Republic of Serbia, Maksima Gorkog 30,
21000 Novi Sad, Serbia; petar.mitrovic@nsseme.com
4Faculty of Agriculture, University of Novi Sad, Trg Dositeja Obradovi´ca 8, 21000 Novi Sad, Serbia;
renatailicic@gmail.com
5Faculty of Biology, University of Belgrade, Studentski Trg 16, 11000 Belgrade, Serbia; slavisas@bio.bg.ac.rs
6Institute for Plant Protection and Environment, Teodora Drajzera 9, 11040 Belgrade, Serbia
*Correspondence: tanjaizbis@gmail.com
Abstract:
The aim of this study was to establish a link between genetic diversity and the geographic
origin of Pectobacterium strains belonging to three species—P. carotovorum,P. versatile, and P. odor-
iferum—isolated from cabbage in Serbia by comparing their sequences with those of strains sourced
from different hosts and countries in Europe, Asia, and North America. Phylogeographic relatedness
was reconstructed using the Templeton, Crandall, and Sing’s (TCS) haplotype network based on
concatenated sequences of the housekeeping genes dnaX,icdA,mdh, and proA, while pairwise genetic
distances were computed by applying the p-distance model. The obtained TCS haplotype networks
indicated the existence of high intra-species genetic diversity among strains of all three species, as
reflected in the 0.2–2.3%, 0.2–2.5%, and 0.1–1.7% genetic distance ranges obtained for P. carotovorum,
P. versatile, and P. odoriferum, respectively. Five new haplotypes (denoted as HPc1–HPc5) were de-
tected among cabbage strains of P. carotovorum, while one new haplotype was identified for both P.
versatile (HPv1) and P. odoriferum (HPo1). None of the TCS haplotype networks provided evidence of
significant correlation between geographic origin and the determined haplotypes, i.e., the infection
origin. However, as haplotype network results are affected by the availability of sequencing data in
public databases for the used genes and the number of analyzed strains, these findings may also be
influenced by small sample size.
Keywords: phylogeographic analysis; TCS haplotype network; Pectobacterium; cabbage
1. Introduction
Plant pathogenic bacteria from the genus Pectobacterium (fam. Pectobacteriaceae) cause
disease symptoms (e.g., soft rot, wilt, and blackleg) on a wide range of angiosperm plant
species, including economically important crops (e.g., potato, tomato, cabbage) grown in ge-
ographically diverse regions (ranging from those with temperate to tropical
climates) [1,2].
Their potential for long-distance dissemination is related to the ability to colonize host
plants from various environmental sources, including soil, aerosols, irrigation water,
groundwater, rainwater, non-host plants, and insects in the vicinity of arable land, as
well as from some remote sources, such as winter mountain snow, waterfalls, rivers, seas,
oceans, etc. [
3
]. Aside from its wide host range, the genus Pectobacterium is known for its
high genetic heterogeneity, both within and between species [
4
]. While this aspect was
previously insufficiently investigated, prompted by the development of new molecular
Microorganisms 2023,11, 2122. https://doi.org/10.3390/microorganisms11082122 https://www.mdpi.com/journal/microorganisms
Microorganisms 2023,11, 2122 2 of 13
tools and advanced techniques for assessing bacterial genetic diversity and phylogeny,
the taxonomy of the genus Pectobacterium has recently received renewed research interest,
resulting in the description of 20 species thus far [5–7].
Host adaptation/specialization, as well as horizontal gene transfer that enables Pecto-
bacterium species to exploit distinct ecological niches and adapt to environmental changes,
are considered the main drivers of their evolution [
6
,
8
]. Accordingly, comparison of their
DNA sequences is the most reliable way to quantify genetic variations (e.g., single nu-
cleotide polymorphism, haplotype structure, synonymous and non-synonymous changes,
recombination events, etc.) within and between natural populations [
9
,
10
]. The findings
yielded by the analysis and comparison of DNA sequences of individuals coexisting within
the same population or those of different populations allow us to address additional ques-
tions regarding the ongoing microevolutionary processes related to population structure,
gene flow, past demographic bottlenecks, and expansions, or geographical colonization
events, etc. [
11
]. However, such investigations require assessment of many strains that
originate from different countries and hosts. Given that changes in metabolic genes may be
related to the adaptation of strains to specific environmental niches and host plants, typing
and analysis of protein-coding loci (multilocus sequence typing and analysis, MLST/MLSA)
can provide sufficient data for distinguishing closely related species [
12
,
13
]. Moreover,
since the process required for obtaining strains from culture collections is often prohibitively
expensive, and permits for some bacterial pathogens can be difficult to acquire, multilocus
phylogenetic analysis can be a viable alternative, given that it exploits the information that
can be derived from genomic sequences that are already deposited in the public databases
[e.g., the National Center for Biotechnology Information (NCBI) database and the Plant
Associated and Environmental Microbes Database (PAMDB)]. Its outcomes would improve
the current understanding of phylogeographic and evolutionary patterns, which may pro-
vide insight into the transmission routes (i.e., whether outbreaks have a common source) as
well as enable the reconstruction of the evolutionary history [
14
]. The Population Analysis
with Reticulate Trees (PopART) software can use such data as input for the analysis of the
available population genetic data and for constructing popular haplotype networks such as
Templeton, Crandall, and Sing’s (TCS), minimum spanning networks (MSNs), and median-
joining networks (MJNs), thus helping visualize intra-species genealogical relationships as
well as advance our knowledge of biogeography and the history of populations [
15
]. The
use of TCS haplotype networks for the determination of phylogeography of other soft-rot-
causing bacteria (e.g., P. brasiliense and Dickeya dianthicola) was previously demonstrated in
the work performed by Markovi´c et al. [
16
] on strains isolated from potato in Serbia, based
on concatenated sequences of four housekeeping genes (acnA,icdA,gapA, and mdh). Thus
far, the TCS algorithm has been successfully applied for providing a phylogeographic in-
sight into the population diversity of bacterial species belonging to different genera, such as
Xanthomonas [17,18], Pseudomonas [19], Agrobacterium [20], Ralstonia [21], and Clavibacter [22].
In Serbia, different Pectobacterium spp.—P. atrosepticum,P. brasiliense,P. carotovorum,
P. odoriferum
,P. punjabense,P. zantedeschiae, and P. versatile—have been isolated from various
hosts [
13
,
16
,
23
–
30
]. Three of these species (P. carotovorum,P. odoriferum, and P. versatile) were
recently described as causal agents of soft rot in cabbage in this region [
13
]. To substantiate
the findings presented in this unique report, in the present study, we aimed to conduct
more extensive research on their genetic heterogeneity, as well as their spread routes and
origin by performing phylogeographic analysis.
2. Materials and Methods
2.1. Sequences of the Pectobacterium spp. Strains Used for Phylogeographic Analysis
For the purpose of this investigation, the sequences of seven Pectobacterium spp. strains,
namely P. carotovorum (Pc2321, Pc3821, Pc4821, Pc5421, and Pc8321), P. odoriferum (Po7521),
and P. versatile (Pv6321), obtained from two cabbage hybrids [Cheers F1 (Takii Seed) and
Hippo F1 (Sakata Seed)] grown in Futog (Vojvodina, Serbia) in 2021 were retrieved from
the NCBI database. These seven Serbian cabbage strains were considered representative of
Microorganisms 2023,11, 2122 3 of 13
each species based on their phenotypic and genotypic features determined previously by
Jeluši´c et al. [13].
In order to reconstruct their phylogeographic relatedness, sequences of
other 19 Pectobacterium spp. strains [P. carotovorum (ATCC 15713, 25.1, WPP14, BP201601.1,
JR1.1, XP-13, and Pcc2520), P. odoriferum (BC S7, JK2.1, and CFBP 1878), and P. versatile (14A,
3-2, SCC1, F131, DSM 30169, MYP201603, SR1, SR12, and Pv1520)] obtained from different
hosts (cabbage, carrot, Chinese cabbage, chicory, coleslaw, cucumber, kimchi cabbage,
potato, and radish) and countries [Europe (Belarus, Denmark, Finland, France, Germany,
Russia, and Serbia), Asia (China and Korea), and North America (USA)] were included in
the analysis. The work performed as a part of this study is therefore a continuation of the
previously conducted and published classical phylogenetic analysis based on the concatenated
sequences of four housekeeping genes [dnaX (DNA polymerase III subunit tau), icdA (isoci-
trate dehydrogenase), mdh (malate dehydrogenase), and proA (gamma-glutamyl phosphate
reductase)], for which the same tested and reference strains were used [
13
]. The maximal
number of strains for comparative analysis was selected for each species in accordance with
the availability of sequences for the four utilized housekeeping genes in the NCBI database.
In the selection of genes for creating TCS haplotype networks, the appropriate sequence
length and good discriminatory ability, as determined during previous work on molecular
characterization of Pectobacterium spp. [13], served as the main criteria.
2.2. Phylogeographic Analysis
Phylogeographic relatedness of the Serbian cabbage strains belonging to three species—
P. carotovorum,P. odoriferum, and P. versatile—was reconstructed using the TCS haplotype
network [
31
]. TCS haplotype networks were generated for each species separately, based
on partial concatenated sequences (1639 nt) of four housekeeping genes (dnaX,icdA,mdh,
and proA), proposed by Sławiak et al. [
32
], Moleleki et al. [
33
], and Ma et al. [
34
]. Sequences
were aligned using the ClustalW Multiple alignment function [
35
] of the BioEdit sequence
alignment editor (v 7.2). Prior to the TCS network construction, DnaSP software v6 [
9
]
was utilized to evaluate DNA polymorphism between the tested strains, as this approach
allowed us to determine the maximal number of haplotypes present within tested strains
isolated in different countries that served as inputs for the construction of TCS haplotype
networks. As a part of the present study, the required TCS haplotype networks were generated
using the TCS algorithm [
36
] implemented in the PopART v. 1.7 program [
15
]. Each circle
on the TCS haplotype network represents one haplotype, while countries from which the
reference strains used for comparative analysis originate are denoted by different colors. The
number of hatch marks along the lines connecting haplotypes indicates the number of nu-
cleotide differences (mutations) detected between those haplotypes. Further, the distribution
of haplotypes by country/continent was graphically presented on the world map generated
in the PopART program, whereby each differently colored circle signifies one haplotype.
Finally, pairwise genetic distances for the concatenated sequences of the seven tested
and nineteen reference Pectobacterium spp. strains were computed in Mega software version
7.0, using the p-distance model/method. Genetic distances were computed for each species
separately and standard errors (SE) were obtained by a bootstrap method with
1000 replicates.
3. Results and Discussion
Phylogeographic Analysis
The TCS haplotype network shown in Figure 1a was constructed based on the concate-
nated sequences of genes dnaX,icdA,mdh, and proA for the five tested Serbian P. carotovorum
strains (Pc2321, Pc3821, Pc4821, Pc5421, and Pc8321) and seven reference P. carotovorum
strains obtained from Belarus (25.1), Denmark (ATCC 15713), Serbia (Pcc2520), China
(XP-13), Korea (BP201601.1 and JR1.1), and the USA (WPP14). As can be seen from the
graph, each of the 12 tested P. carotovorum strains formed a single haplotype (designated as
HPc1–HPc12). Based on their relatedness, haplotypes were divided into three major genetic
clades/haplogroups (I–III) of the TCS haplotype network. Four of the six P. carotovorum
strains/haplotypes placed within clade I were isolated in Europe (HPc5–HPc7, HPc9),
Microorganisms 2023,11, 2122 4 of 13
while the remaining two were isolated in North America (HPc8) and Asia (HPc12), each
exhibiting greater similarity with other clade I members relative to the strains placed into
other two haplogroups (II and III). The centrally positioned haplogroup II consisted of only
two Serbian P. carotovorum strains isolated from cabbage, Pc2321 (HPc1) and Pc4821 (HPc3),
which are the most closely related to the ancestral vector (marked with an arrow), differ-
ing in three and two nucleotides, respectively. Finally, clade III included four
P. carotovorum
strains/haplotypes, two of which originated from Europe and were isolated from cabbage
grown in Serbia [Pc3821 (HPc2) and Pc5421 (HPc4)], and two originated from Asia, and were
isolated from radish [JR1.1 (HPc11)] in Korea and from potato in China [XP-13 (HPc10)]. As
shown on the world map (Figure 1b) depicting all twelve haplotypes detected within the
tested strains, six (HPc1–HPc6) were identified in Serbia, two (HPc11 and HPc12) in Korea,
and one each in Denmark (HPc7), Belarus (HPc9), USA (HPc8), and China (HPc10).
The obtained TCS haplotype network clearly indicates the existence of a remarkable
intra-species genetic heterogeneity within the tested and reference P. carotovorum strains.
As specified above, while twelve haplotypes (HPc1–HPc12) were determined for strains
originating from three continents (Europe, Asia, and North America) and from four hosts
(cabbage, potato, cucumber, and radish), five of these haplotypes (HPc1–HPc5) were
distinguished for the five Serbian cabbage strains (Pc2321, Pc3821, Pc4821, Pc5421, and
Pc8321). The same five cabbage strains were separated into four clusters (I: Pc2321 and
Pc4821, II: Pc3821, III: Pc5421, and IV: Pc8321) of the neighbor-joining phylogenetic tree
previously generated by Jeluši´c et al. [
13
], thus confirming the existence of a complex
population structure within the Serbian P. carotovorum strains isolated from this host.
Population complexity is also reflected in the existence of four (HPc1–HPc4) P. carotovorum
genotypes in a single cabbage field measuring only 0.5 hectares in size (field I—cabbage
hybrid Cheers F1) [
13
]. However, based on the obtained TCS haplotype network, we
cannot ascertain if there is a link between geographic origin and/or host of isolation
and the determined haplotypes or make any assumptions regarding the infection origin.
Nevertheless, it is important to emphasize that the reliability of phylogeny and haplotype
networks is affected by the sample size, i.e., the number of strains included in the study,
which depends on their availability in public databases as well as the choice of genes
included in the research and their discriminatory ability. Despite the limitations imposed
by the sample size, the obtained TCS haplotype network provides valuable information on
the population structure of P. carotovorum in Serbia and other countries (Belarus, Denmark,
China, Korea, and the USA) included in the analysis. Its benefit is further increased by the
fact that, to the best of our knowledge, this is a pioneering study using the TCS haplotype
network for exploring the phylogeography of P. carotovorum, as well as P. versatile and P.
odoriferum. Thus far, only P. brasiliense strains from the Pectobacterium genus have been
subjected to such analysis, which was based on a different combination of acnA,gapA, icdA,
and mdh housekeeping genes [
16
]. The obtained findings indicate the existence of four
haplotypes (PCB-1, PCB-2, PCB-3, and PCB-4) among twenty tested Serbian P. brasiliense
strains from potato; however, no connection between the geographic origin and the genetic
diversity was established [
16
]. The detection of a pronounced genetic diversity among P.
carotovorum strains in this study is not surprising, given that it was previously confirmed
by several authors. For instance, Gallelli et al. [
37
] indicated the existence of 14 haplotypes
among 24 P. carotovorum strains isolated from artichoke in southern Italy (Sele valley,
Campania), based on DNA profiling methods (repetitive-sequence-based PCR and M13-
PCR). In the study conducted by Alvarado and colleagues, 39 tested P. carotovorum isolates
collected from Chinese cabbage in north-eastern Brazil were shown to be polymorphic and
were separated into 32 groups, also based on the repetitive-sequence-based PCR with REP-,
ERIC-, and BOX-PCR primers [
38
]. According to Nabhan et al. [
39
], MLSA involving seven
housekeeping genes (acnA,gapA,proA,icdA,mtlD,mdh, and pgi) enabled separation of sixty-
three strains belonging to the P. carotovorum complex (including subspecies carotovorum,
odoriferum, and brasiliensis based on earlier taxonomy), isolated from various hosts and
countries, into five genetic clusters (PcI–PcV), three of which (PcI, PcII, and PcV) belonged
Microorganisms 2023,11, 2122 5 of 13
to P. carotovorum subsp. carotovorum strains. However, these authors also failed to establish
any correlation between the geographic origin and/or host affiliation and genotype [39].
Microorganisms 2023, 11, x FOR PEER REVIEW 5 of 14
ing methods (repetitive-sequence-based PCR and M13-PCR). In the study conducted by
Alvarado and colleagues, 39 tested P. carotovorum isolates collected from Chinese cabbage
in north-eastern Brazil were shown to be polymorphic and were separated into 32
groups, also based on the repetitive-sequence-based PCR with REP-, ERIC-, and
BOX-PCR primers [38]. According to Nabhan et al. [39], MLSA involving seven house-
keeping genes (acnA, gapA, proA, icdA, mtlD, mdh, and pgi) enabled separation of six-
ty-three strains belonging to the P. carotovorum complex (including subspecies caroto-
vorum, odoriferum, and brasiliensis based on earlier taxonomy), isolated from various hosts
and countries, into five genetic clusters (PcI–PcV), three of which (PcI, PcII, and PcV)
belonged to P. carotovorum subsp. carotovorum strains. However, these authors also failed
to establish any correlation between the geographic origin and/or host affiliation and
genotype [39].
Figure 1. (a) Templeton, Crandall, and Sing’s (TCS) haplotype network showing the phylogeo-
graphic position of the five tested Serbian P. carotovorum strains (Pc2321, Pc3821, Pc4821, Pc5421,
and Pc8321) and seven reference P. carotovorum strains (ATCC 15713, 25.1, WPP14, BP201601.1,
JR1.1, XP-13, and Pcc2520). Different colors on the TCS network represent countries in which the
Figure 1.
(
a
) Templeton, Crandall, and Sing’s (TCS) haplotype network showing the phylogeographic
position of the five tested Serbian P. carotovorum strains (Pc2321, Pc3821, Pc4821, Pc5421, and Pc8321)
and seven reference P. carotovorum strains (ATCC 15713, 25.1, WPP14, BP201601.1, JR1.1, XP-13, and
Pcc2520). Different colors on the TCS network represent countries in which the tested/reference
strains were isolated. The number of hatch marks reflects the number of nucleotide differences
detected between haplotypes, while the arrow points to an ancestral genotype; (
b
) World map
showing the distribution of the 12 detected haplotypes (HPc1–HPc12) of the tested and reference
P. carotovorum strains by country.
The pairwise genetic distances between the tested and reference P. carotovorum strains
calculated as a part of the present study shown in Table 1provide support for the distri-
bution of haplotypes on the TCS network. Genetic distances between the 12 compared
P. carotovorum
strains ranged from 0.2% (between Serbian strains Pc2321 and Pc4821 from
cabbage) to 2.3% (between Serbian strains Pcc2520 and Pc3821 from potato and cabbage,
Microorganisms 2023,11, 2122 6 of 13
respectively). Similar results were obtained for the five tested Serbian P. carotovorum strains
from cabbage, as the estimated distance ranged from 0.2% (between strains Pc2321 and
Pc4821) to 2.1% (between strains Pc5421 and Pc8321).
Table 1.
Pairwise genetic distances (p-distance model/method) and standard errors (SE) between
tested and reference P. carotovorum strains, calculated based on the partial concatenated sequences of
genes dnaX,icdA,mdh, and proA.
P. carotovorum
Strains
p-Distance/SE *
123456789101112
1. Pc2321 0.003 0.001 0.003 0.003 0.003 0.004 0.003 0.003 0.002 0.003 0.002
2. Pc3821 0.015 0.003 0.003 0.003 0.004 0.003 0.003 0.003 0.002 0.003 0.003
3. Pc4821 0.002 0.014 0.003 0.003 0.003 0.004 0.003 0.003 0.002 0.003 0.002
4. Pc5421 0.016 0.015 0.015 0.004 0.004 0.004 0.004 0.003 0.003 0.003 0.004
5. Pc8321 0.015 0.015 0.015 0.021 0.003 0.002 0.002 0.003 0.003 0.003 0.003
6. Pcc2520 0.016 0.023 0.016 0.021 0.016 0.004 0.003 0.002 0.003 0.003 0.002
7. ATCC 15713 0.021 0.020 0.020 0.020 0.009 0.020 0.003 0.003 0.003 0.003 0.003
8. WPP14 0.015 0.013 0.014 0.018 0.005 0.015 0.013 0.003 0.003 0.003 0.003
9. 25.1 0.019 0.019 0.018 0.018 0.013 0.009 0.018 0.014 0.003 0.003 0.003
10. XP-13 0.011 0.009 0.010 0.015 0.014 0.020 0.017 0.012 0.021 0.003 0.003
11. JR1.1 0.012 0.013 0.012 0.013 0.015 0.020 0.017 0.015 0.019 0.015 0.003
12. BP201601.1 0.009 0.016 0.009 0.020 0.012 0.010 0.015 0.012 0.012 0.016 0.015
*Standard Error.
Similar findings were reported by Nabhan et al. [
39
], who calculated a 4.0% average
genetic distance among P. carotovorum strains obtained from potato in Syria based on the
sequences of seven housekeeping genes. Among all tested and reference P. carotovorum
strains examined as part of the present study, Serbian strains from cabbage [Pc2321 (2.0%)
and Pc4821 (2.1%)] were the most distant from the P. carotovorum type strain ATCC 15713
isolated from potato in Denmark, while the remaining three Serbian cabbage strains [Pc3821
(2.3%), Pc5421 (2.1%), and Pc8321 (1.6%)] were located furthest away from strain Pcc2520
isolated from potato in Serbia. These results confirm a highly complex and polymorphic
P. carotovorum
population structure in Serbia, irrespective of host type. The greatest simi-
larity between strains Pc2321 (HPc1) and Pc4821 (HPc3) is also reflected in their grouping
within the same cluster (haplogroup II) on the TCS haplotype network. Their percent
identity with the strains deposited into the NCBI database ranged from 99.75% (mdh) to
100% (dnaX,icdA, or proA) depending on the used gene [
13
]. According to the previously
performed phylogenetic analysis based on concatenated sequences [
13
] and the TCS net-
work obtained in this study, cabbage strain Pc5421 (HPc4, haplogroup III) was the most
closely related to the radish strain JR1.1 isolated from Korea (genetic distance 1.3%), while
being the most divergent from the ancestral vector (differing in 23 nucleotides). The percent
similarity of this strain with the strains deposited in the NCBI database ranged from 97.76%
(proA) to 99.44% (icdA) [
13
]. Conversely, the strain Pc8321 (HPc5, haplogroup I) obtained
from another cabbage hybrid (Hippo F1) and from another tested field (field II) was found
to be the most closely related to the P. carotovorum type strain ATCC 15713 (genetic distance
0.9%). It also shared 97.91% (proA) to 100% (dnaX,icdA, and mdh) identity with the strains
sourced from the NCBI database [
13
]. Although the P. carotovorum strains found in field II
(1 ha in size) were genetically homogeneous in terms of the existence of only one haplotype
(HPc5), while the remaining four (HPc1–HPc4) were detected on cabbage hybrid Cheers F1
(field I), it is important to emphasize that this cabbage hybrid harbored three pathogenic
Pectobacterium spp. (P. carotovorum,P. versatile, and P. odoriferum) [13].
Figure 2a shows the TCS haplotype network generated for the 10 P. versatile strains
[tested (Pv6321) and reference strains (14A, 3-2, SCC1, F131, DSM 30169, MYP201603, SR1,
SR12, and Pv1520)]. These strains were separated into ten haplotypes (designated as HPv1–
HPv10) that formed two clades/haplogroups (I and II) within the TCS network. The tested
Serbian P. versatile strain Pv6321 (HPv1) from cabbage was placed within the haplogroup
I
,
Microorganisms 2023,11, 2122 7 of 13
together with the five reference P. versatile strains, three of which were isolated from potato
in Belarus (3-2, HPv5), Russia (F131, HPv7), and Korea (MYP201603, HPv8), one from
cabbage in Germany (DSM 30169, HPv6), and one from carrot in the USA (SR1, HPv9). The
P. versatile strain isolated from potato in Serbia (Pv1520, HPv2) was placed in haplogroup II
with the strains isolated from potato in Finland (SCC1, HPv3) and Belarus (14A, HPv4),
as well as from coleslaw in the USA (SR12, HPv10). Based on the presented world map
(Figure 2b), two haplotypes were detected in Serbia (HPv1 and HPv2), Belarus (HPv4 and
HPv5), and the USA (HPv9 and HPv10), while one haplotype was detected in Finland
(HPv3), Germany (HPv6), Russia (HPv7), and Korea (HPv8).
Microorganisms 2023, 11, x FOR PEER REVIEW 9 of 14
Figure 2. (a) Templeton, Crandall, and Sing’s (TCS) haplotype network showing phylogeographic
position of one tested Serbian P. versatile (Pv6321) strain and nine reference P. versatile strains (14A,
3-2, SCC1, F131, DSM 30169, MYP201603, SR1, SR12, and Pv1520). Different colors on the TCS
network represent countries in which the tested/reference strains were isolated. The number of
hatch marks reflects the number of nucleotide differences detected between haplotypes; (b) World
map showing the distribution of the 10 detected haplotypes (HPv1–HPc10) of the tested and ref-
erence P. versatile strains by country.
Figure 2.
(
a
) Templeton, Crandall, and Sing’s (TCS) haplotype network showing phylogeographic
position of one tested Serbian P. versatile (Pv6321) strain and nine reference P. versatile strains (14A, 3-2,
SCC1, F131, DSM 30169, MYP201603, SR1, SR12, and Pv1520). Different colors on the TCS network
represent countries in which the tested/reference strains were isolated. The number of hatch marks
reflects the number of nucleotide differences detected between haplotypes; (b) World map showing
the distribution of the 10 detected haplotypes (HPv1–HPc10) of the tested and reference P. versatile
strains by country.
Microorganisms 2023,11, 2122 8 of 13
Similar to the results reported for P. carotovorum strains, Serbian P. versatile cabbage
strain Pv6321 formed a new haplotype (HPv1), which markedly differed from the haplo-
types (HPv2–HPv10) detected for the reference P. versatile strains. As can be seen from
Table 2, this strain was the most closely related to the German strain DSM 30169, which
also originated from cabbage, differing in five nucleotides (p-distance 0.2%). With the ex-
ception of this relationship, the TCS network did not indicate any type of phylogeographic
correlation between the tested and reference P. versatile strains. On the other hand, based
on the calculated pairwise genetic distances, P. versatile strain Pv6321 was the most distant
(differing in 39 nucleotides, p-distance 1.9%) from the strain MYP201603 isolated from
potato in Korea (Table 2).
Table 2.
Pairwise genetic distances (p-distance model/method) and standard errors (SE) between
tested and reference P. versatile strains, calculated based on the partial concatenated sequences of
genes dnaX,icdA,mdh, and proA.
P. versatile
Strains
p-Distance/SE *
12345678910
1. Pv6321 0.003 0.003 0.001 0.004 0.003 0.003 0.003 0.003 0.003
2. Pv1520 0.016 0.002 0.003 0.003 0.004 0.003 0.003 0.001 0.003
3. F131 0.013 0.004 0.003 0.003 0.004 0.003 0.003 0.001 0.003
4. DSM 30169 0.002 0.015 0.013 0.004 0.003 0.003 0.003 0.003 0.003
5. MYP201603 0.019 0.012 0.010 0.019 0.004 0.004 0.004 0.003 0.003
6. SR1 0.018 0.020 0.023 0.017 0.023 0.004 0.003 0.004 0.003
7. SR12 0.018 0.015 0.014 0.016 0.022 0.025 0.003 0.003 0.003
8. 14A 0.016 0.015 0.015 0.015 0.021 0.021 0.013 0.003 0.004
9. SCC1 0.015 0.002 0.003 0.013 0.013 0.022 0.013 0.014 0.003
10. 3-2 0.015 0.015 0.015 0.012 0.020 0.017 0.019 0.021 0.016
*Standard Error.
The results yielded by examining the TCS haplotype network generated as a part of this
study are in accordance with the phylogenetic tree previously obtained by Jeluši´c et al. [
13
].
The detection of 10 haplotypes for P. versatile strains with genetic distances ranging from
0.2% to 2.5% (Table 2) is indicative of a pronounced genetic polymorphism within the
P. versatile species. In accordance with the number of strains from the three continents
included in the comparison, seven P. versatile haplotypes were detected in Europe (HPv1–
HPv7), two in the USA (HPv9 and HPv10), and one in Asia (HPv8). However, these results
are completely dependent on the sample size, which is affected by (i) the number of publicly
available strains (which can be limited due to the recent description of certain species),
as well as (ii) the number and (iii) the combination of genes included in the comparison.
Nonetheless, they concur with the report published by Ma and colleagues on a complex
population structure of P. versatile in the northeastern United States based on the dnaX
gene sequences [
40
]. The same authors indicated a much greater prevalence of P. versatile
compared to other Pectobacterium spp. identified in this region, and ascribed this disparity
to its better fitness and thus greater resilience to environmental conditions and/or cultivar
characteristics. Given that P. versatile was recently described as a pathogen on cabbage in
Serbia [
13
] and that no information regarding its geographic origin is currently available, it
can be speculated that this species diverged over time from the dominant P. carotovorum
populations through the accumulation of mutations as a result of the section pressure
related to host, cultivar, or other pertinent factors. It is context, it is also worth noting that
Park et al. [
41
] characterized the new P. versatile strain KNUB-02-21 on kimchi cabbage in
Korea based on genes dnaX,leuS, and recA. These authors pointed to the existence of intra-
species genetic heterogeneity among P. versatile strains (tested and reference), revealing
three different genotypes among five compared strains. Based on three housekeeping genes
(dnaX,leuS, and gapA), eight P. versatile strains isolated from cabbage (CaKh26, CaKh54,
Microorganisms 2023,11, 2122 9 of 13
CaKh77, and CaKh83) and potato (PH35, PH47, PH62, and PH75) in Iran were divided into
two clusters within the phylogenetic tree, each corresponding to one host [42].
As can be seen from Figure 3a, four detected haplotypes (designated as HPo1–HPo4)
of the four tested (Po7521) and reference (BC S7, JK2.1, and CFBP 1878) P. odoriferum strains
were divided within the TCS haplotype network into three clades/haplogroups (I–III) in
relation to the ancestral vector (marked with an arrow), which occupied a central position.
The Serbian P. odoriferum strain from cabbage Po7521 (HPo1) was placed within clade II,
together with the type P. odoriferum strain CFBP 1878 (HPo2) isolated from chicory in France,
from which it differed in one nucleotide only. Haplotypes of the reference
P. odoriferum
strains from Asia, isolated in Korea (JK2.1, HPo3) and China (BC S7, HPo4), were placed
in clade II and III, respectively. Strain Po7521 differed from the ancestral vector in two
nucleotides, while having only one different nucleotide relative to strains CFBP 1878 and BC
S7. On the other hand, the strain JK2.1 differed from the ancestral vector in 26 nucleotides.
In summary, as shown on the world map (Figure 3b), one haplotype was detected in each
country—Serbia (HPo1), France (HPo2), Korea (HPo3), and China (HPo4).
Microorganisms 2023, 11, x FOR PEER REVIEW 10 of 14
Figure 3. (a) Templeton, Crandall, and Sing’s (TCS) haplotype network showing the phylogeo-
graphic position of one tested Serbian P. odoriferum (Po7521) strain and three reference P. odoriferum
strains (BC S7, JK2.1, and CFBP 1878). Different colors on the TCS network represent countries in
which the tested/reference strains were isolated. The number of hatch marks denotes the number of
nucleotide differences detected between haplotypes, while the arrow points to an ancestral geno-
type; (b) World map showing the distribution of the four detected haplotypes (HPo1–HPo4) of the
tested and reference P. odoriferum strains by country.
Based on the TCS haplotype network constructed with tested and reference strains,
a new haplotype of P. odoriferum (HPo1) was determined for the Serbian cabbage strain
Po7521. The observed haplotype was the most similar to one of the P. odoriferum type
strains [CFBP 1878 (HPo2)] isolated from chicory in France, differing in only one nucleo-
tide (p-distance 0.1%, Table 3). On the other hand, it differed the most (in 28 nucleotides,
Figure 3a) from the strain JK2.1 obtained from kimchi cabbage in Korea (p-distance 1.7%,
Tabl e 3). According to Jelušić et al. [13], the percent identity of Po7521 with the strains
sourced from the NCBI database ranged from 99.57% (proA) to 100% (icdA and mdh),
depending on the considered gene.
Figure 3.
(
a
) Templeton, Crandall, and Sing’s (TCS) haplotype network showing the phylogeographic
position of one tested Serbian P. odoriferum (Po7521) strain and three reference P. odoriferum strains
(BC S7, JK2.1, and CFBP 1878). Different colors on the TCS network represent countries in which
the tested/reference strains were isolated. The number of hatch marks denotes the number of
nucleotide differences detected between haplotypes, while the arrow points to an ancestral genotype;
(
b
) World map showing the distribution of the four detected haplotypes (HPo1–HPo4) of the tested
and reference P. odoriferum strains by country.
Microorganisms 2023,11, 2122 10 of 13
Based on the TCS haplotype network constructed with tested and reference strains,
a new haplotype of P. odoriferum (HPo1) was determined for the Serbian cabbage strain
Po7521. The observed haplotype was the most similar to one of the P. odoriferum type
strains [CFBP 1878 (HPo2)] isolated from chicory in France, differing in only one nucleotide
(
p-distance
0.1%, Table 3). On the other hand, it differed the most (in 28 nucleotides,
Figure 3a) from the strain JK2.1 obtained from kimchi cabbage in Korea (p-distance 1.7%,
Table 3). According to Jeluši´c et al. [
13
], the percent identity of Po7521 with the strains
sourced from the NCBI database ranged from 99.57% (proA) to 100% (icdA and mdh),
depending on the considered gene.
Table 3.
Pairwise genetic distances (p-distance model/method) and standard errors (SE) between
tested and reference P. odoriferum strains, calculated based on the partial concatenated sequences of
genes dnaX,icdA,mdh, and proA.
P. odoriferum Strains p-Distance/SE *
1 2 3 4
1. Po7521 0.001 0.004 0.001
2. BC S7 0.002 0.003 0.001
3. JK2.1 0.017 0.016 0.003
4. CFBP1878 0.001 0.001 0.016
*Standard Error.
Once again, no correlation between geographic origin and/or the host of isolation
could be established based on the constructed TCS network. These findings are supported
by those reported by Oskiera et al., who determined the existence of intra-species genetic
heterogeneity among P. carotovorum subsp. odoriferum strains isolated from cabbage and
Chinese cabbage in Poland based on BOX- and ERIC-PCR, showing three and four distinct
DNA fingerprinting patterns, respectively [
43
]. The genetic polymorphism among these
strains was further confirmed based on the sequences of the 16S rRNA gene and five
housekeeping genes (gyrB,infB,rpoB,atpD, and rpoS) [43].
Even though the TCS analysis performed in the present study focused on P. odoriferum
and incorporated only a few strains, due to the recent description of this species and the
scarcity of publicly available genomic data, the results reported in this work undoubtedly
shed light on the issue of the phylogeography of the Pectobacterium genus. They also allow
us to speculate that the two recently isolated species, P. versatile and P. odoriferum, could
have been previously present on cabbage in Serbia, but due to their recent description and
separation from the P. carotovorum group, they were not established in previous studies.
We further posit that the observed intra-species genetic heterogeneity among cabbage
strains might have arisen during the dynamic remodeling of genome content (gain, loss,
duplication, and transfer of genes) which would have accelerated the evolution of the
genus Pectobacterium [44].
4. Conclusions
TCS haplotype networks comprising sequences of P. carotovorum, P. versatile, and
P. odoriferum
strains isolated from cabbage in Serbia indicate high intra-species diversity
among all three bacterial species. The results yielded by this study reveal five new hap-
lotypes among P. carotovorum strains (HPc1–HPc5) and one new haplotype for both the
P. versatile
(HPv1) and P. odoriferum (HPo1) strains. These results provide further evidence
of the usefulness of this approach in revealing closely related but different bacterial lineages
that belong to the same species and that have been isolated from the same host plant
in the same country. However, none of the TCS haplotype networks showed a correla-
tion between geographic origin and the determined haplotypes among analyzed cabbage
Pectobacterium strains.
Microorganisms 2023,11, 2122 11 of 13
Author Contributions:
Conceptualization, A.J. and T.P.M.; methodology, A.J. and S.M.; software, A.J.
and S.M.; validation, A.J., S.M. and R.I.; formal analysis, A.J.; investigation, A.J. and T.P.M.; resources,
A.J., P.M. and T.P.M.; data curation, A.J. and T.P.M.; writing—original draft preparation, A.J., T.P.M.,
M.S., R.I. and S.S.; writing—review and editing, A.J., T.P.M., M.S., R.I. and S.S.; visualization, A.J.,
T.P.M., M.S., R.I., S.S., P.M. and S.M.; supervision, T.P.M. and M.S. All authors have read and agreed
to the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: Not applicable.
Acknowledgments:
This research was supported by the Ministry of Education, Science and Tech-
nological Development of the Republic of Serbia, contract Nos. 451-03-47/2023-01/200053, 451-
03-47/2023-01/200032, 451-03-47/2023-01/200117, 451-03-47/2023-01/200178, and 451-03-47/2023-
01/200010.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
References
1.
Reverchon, S.; Muskhelisvili, G.; Nasser, W. Virulence program of a bacterial plant pathogen: The Dickeya model. Prog. Mol. Biol.
Transl. Sci. 2016,142, 51–92. [CrossRef] [PubMed]
2.
Amir, R.; Maqsood, W.; Munir, F.; Fatima, N.; Siddiqa, A.; Ahmad, J. Pan-genomics of plant pathogens and its applications. In
Pan-Genomics: Applications, Challenges, and Future Prospects; Barh, D., Soares, S., Tiwari, S., Azevedo, V., Eds.; Academic Press:
Cambridge, MA, USA, 2020; pp. 121–145. [CrossRef]
3.
Toth, I.K.; Barny, M.; Brurberg, M.B.; Condemine, G.; Czajkowski, R.; Elphinstone, J.G.; Helias, V.; Johnson, S.B.; Moleleki, L.N.;
Pirhonen, M.; et al. Pectobacterium and Dickeya: Environment to disease development. In Plant Diseases Caused by Dickeya and
Pectobacterium Species; Van Gijsegem, F., van der Wolf, J.M., Toth, I.K., Eds.; Springer: Cham, Switzerland, 2021; pp. 39–84.
[CrossRef]
4.
Xu, P.; Wang, H.; Qin, C.; Li, Z.; Lin, C.; Liu, W.; Miao, W. Analysis of the taxonomy and pathogenic factors of Pectobacterium
aroidearum L6 using whole-genome sequencing and comparative genomics. Front. Microbiol.
2021
,12, 679102. [CrossRef]
[PubMed]
5.
Pasanen, M.; Waleron, M.; Schott, T.; Cleenwerck, I.; Misztak, A.; Waleron, K.; Pritchard, L.; Bakr, R.; Degefu, Y.; van der Wolf, J.;
et al. Pectobacterium parvum sp. nov., having a Salmonella SPI-1-like Type III secretion system and low virulence. Int. J. Syst. Evol.
Microbiol. 2020,70, 2440–2448. [CrossRef] [PubMed]
6.
Jonkheer, E.M.; Brankovics, B.; Houwers, I.M.; van der Wolf, J.M.; Bonants, P.J.; Vreeburg, R.A.; Bollema, R.; de Haan, J.R.; Berke,
L.; Smit, S.; et al. The Pectobacterium pangenome, with a focus on Pectobacterium brasiliense, shows a robust core and extensive
exchange of genes from a shared gene pool. BMC Genom. 2021,22, 265. [CrossRef] [PubMed]
7.
Zhou, J.; Hu, M.; Hu, A.; Li, C.; Ren, X.; Tao, M.; Xue, Y.; Chen, S.; Tang, C.; Xu, Y.; et al. Isolation and genome analysis of
Pectobacterium colocasium sp. nov. and Pectobacterium aroidearum, two new pathogens of taro. Front. Plant Sci.
2022
,13, 852750.
[CrossRef]
8.
Khadka, N.; Joshi, J.R.; Reznik, N.; Chriker, N.; Nudel, A.; Zelinger, E.; Kerem, Z.; Yedidia, I. Host specificity and differential
pathogenicity of Pectobacterium strains from dicot and monocot hosts. Microorganisms 2020,8, 1479. [CrossRef]
9.
Rozas, J.; Rozas, R. DnaSP, DNA sequence polymorphism: An interactive program for estimating population genetics parameters
from DNA sequence data. Bioinformatics 1995,11, 621–625. [CrossRef]
10.
Rozas, J.; Sánchez-DelBarrio, J.C.; Messeguer, X.; Rozas, R. DnaSP, DNA polymorphism analyses by the coalescent and other
methods. Bioinformatics 2003,19, 2496–2497. [CrossRef]
11.
Paradis, E. Analysis of haplotype networks: The randomized minimum spanning tree method. Methods Ecol. Evol.
2018
,9,
1308–1317. [CrossRef]
12.
Nabhan, S.; De Boer, S.H.; Maiss, E.; Wydra, K. Taxonomic relatedness between Pectobacterium carotovorum subsp. carotovorum,
Pectobacterium carotovorum subsp. odoriferum and Pectobacterium carotovorum subsp. brasiliense subsp. nov. J. Appl. Microbiol.
2012
,
113, 904–913. [CrossRef]
13.
Jeluši´c, A.; Mitrovi´c, P.; Markovi´c, S.; Iliˇci´c, R.; Milovanovi´c, P.; Stankovi´c, S.; Popovi´c Milovanovi´c, T. Diversity of Bacterial Soft
Rot-Causing Pectobacterium Species Affecting Cabbage in Serbia. Microorganisms 2023,11, 335. [CrossRef] [PubMed]
14.
Achtman, M. Evolution, population structure, and phylogeography of genetically monomorphic bacterial pathogens. Annu. Rev.
Microbiol. 2008,62, 53–70. [CrossRef] [PubMed]
15.
Leigh, J.W.; Bryant, D. POPART: Full-feature software for haplotype network construction. Methods Ecol. Evol.
2015
,6, 1110–1116.
[CrossRef]
Microorganisms 2023,11, 2122 12 of 13
16.
Markovi´c, S.; Stankovi´c, S.; Jeluši´c, A.; Iliˇci´c, R.; Kosovac, A.; Pošti´c, D.; Popovi´c, T. Occurrence and Identification of Pectobacterium
carotovorum subsp. brasiliensis and Dickeya dianthicola Causing Blackleg in some Potato Fields in Serbia. Plant Dis.
2021
,105,
1080–1090. [CrossRef]
17.
Khojasteh, M.; Taghavi, S.M.; Khodaygan, P.; Hamzehzarghani, H.; Chen, G.; Bragard, C.; Koebnik, R.; Osdaghi, E. Molecular
typing reveals high genetic diversity of Xanthomonas translucens strains infecting small-grain cereals in Iran. Appl. Environ.
Microbiol. 2019,85, e01518-19. [CrossRef]
18.
Iliˇci´c, R.; Jeluši´c, A.; Milovanovi´c, P.; Stankovi´c, S.; Zeˇcevi´c, K.; Stanisavljevi´c, R.; Popovi´c, T. Characterization of Xanthomonas
arboricola pv. pruni from Prunus spp. orchards in Western Balkans. Plant Pathol. 2023,72, 290–299. [CrossRef]
19.
Ilici´c, R.; Popovi´c, T.; Markovi´c, S.; Jeluši´c, A.; Bagi, F.; Vlajic, S.; Stankovi´c, S. Genetic diversity of Pseudomonas syringae pv.
syringae isolated from sweet cherry in southern and northern regions in Serbia. Genetika 2021,53, 247–262. [CrossRef]
20.
Mafakheri, H.; Taghavi, S.M.; Puławska, J.; de Lajudie, P.; Lassalle, F.; Osdaghi, E. Two novel genomospecies in the Agrobacterium
tumefaciens species complex associated with rose crown gall. Phytopathology 2019,109, 1859–1868. [CrossRef]
21.
Markovi´c, S.; Stankovi´c, S.; Iliˇci´c, R.; Veljovi´c Jovanovi´c, S.; Mili´c Komi´c, S.; Jeluši´c, A.; Popovi´c, T. Ralstonia solanacearum as
a potato pathogen in Serbia: Characterization of strains and influence on peroxidase activity in tubers. Plant Pathol.
2021
,70,
1945–1959. [CrossRef]
22.
Ansari, M.; Taghavi, S.M.; Hamzehzarghani, H.; Valenzuela, M.; Siri, M.I.; Osdaghi, E. Multiple introductions of tomato pathogen
Clavibacter michiganensis subsp. michiganensis into Iran as revealed by a global-scale phylogeographic analysis. Appl. Environ.
Microbiol. 2019,85, e02098-19. [CrossRef]
23.
Waleron, M.; Misztak, A.; Waleron, M.; Franczuk, M.; Jo´nca, J.; Wielgomas, B.; Mikici´nski, A.; Popovi´c, T.; Waleron, K. Pectobac-
terium zantedeschiae sp. nov. a new species of a soft rot pathogen isolated from Calla lily (Zantedeschia spp.). Syst. Appl. Microbiol.
2019,42, 275–283. [CrossRef] [PubMed]
24.
Markovi´c, S.; Mili´c Komi´c, S.; Jeluši´c, A.; Iliˇci´c, R.; Bagi, F.; Stankovi´c, S.; Popovi´c, T. First report of Pectobacterium versatile causing
blackleg of potato in Serbia. Plant Dis. 2022,106, 312. [CrossRef] [PubMed]
25.
Gavrilovi´c, V.; Obradovi´c, A.; Arsenijevi´c, M. Bacterial soft rot of carrot, parsley and celery. In Plant Pathogenic Bacteria; De Boer,
S.H., Ed.; Springer: Dordrecht, The Netherlands, 2001; pp. 269–271.
26.
Gaši´c, K.; Gavrilovi´c, V.; Dolovac, N.; Trkulja, N.; Živkovi´c, S.; Risti´c, D.; Obradovi´c, A. Pectobacterium carotovorum subsp.
carotovorum-the causal agent of broccoli soft rot in Serbia. Pestic. Phytomedicine 2014,29, 249–255. [CrossRef]
27.
Popovi´c, T.; Jeluši´c, A.; Milovanovi´c, P.; Janjatovi´c, S.; Budnar, M.; Dimki´c, I.; Stankovi´c, S. First report of Pectobacterium
atrosepticum, causing bacterial soft rot on calla lily in Serbia. Plant Dis. 2017,101, 2145. [CrossRef]
28.
Zlatkovi´c, N.; Proki´c, A.; Gaši´c, K.; Kuzmanovi´c, N.; Ivanovi´c, M.; Obradovi´c, A. First report of Pectobacterium carotovorum subsp.
brasiliense causing soft rot on squash and watermelon in Serbia. Plant Dis. 2019,103, 2667. [CrossRef]
29.
Loc, M.; Miloševi´c, D.; Ivanovi´c, Ž.; Ignjatov, M.; Budakov, D.; Grahovac, J.; Grahovac, M. Genetic Diversity of Pectobacterium spp.
on Potato in Serbia. Microorganisms 2022,10, 1840. [CrossRef]
30.
Loc, M.; Miloševi´c, D.; Ignjatov, M.; Ivanovi´c, Ž.; Budakov, D.; Grahovac, J.; Vlajkov, V.; Pajˇcin, I.; Grahovac, M. First report of
Pectobacterium punjabense causing potato soft rot and blackleg in Serbia. Plant Dis. 2022,106, 1513. [CrossRef]
31.
Templeton, A.R.; Crandall, K.A.; Sing, C.F. A cladistic analysis of phenotypic associations with haplotypes inferred from restriction
endonuclease mapping and DNA sequence data. III. Cladogram estimation. Genetics 1992,132, 619–633. [CrossRef]
32.
Sławiak, M.; van Beckhoven, J.R.; Speksnijder, A.G.; Czajkowski, R.; Grabe, G.; van der Wolf, J.M. Biochemical and genetical
analysis reveal a new clade of biovar 3 Dickeya spp. strains isolated from potato in Europe. Eur. J. Plant Pathol.
2009
,125, 245–261.
[CrossRef]
33.
Moleleki, L.N.; Onkendi, E.M.; Mongae, A.; Kubheka, G.C. Characterisation of Pectobacterium wasabiae causing blackleg and soft
rot diseases in South Africa. Eur. J. Plant Pathol. 2013,135, 279–288. [CrossRef]
34.
Ma, B.; Hibbing, M.E.; Kim, H.S.; Reedy, R.M.; Yedidia, I.; Breuer, J.; Charkowski, A.O. Host range and molecular phylogenies of
the soft rot enterobacterial genera Pectobacterium and Dickeya.Phytopathology 2007,97, 1150–1163. [CrossRef] [PubMed]
35.
Thompson, J.D.; Higgins, D.G.; Gibson, T.J. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res.
1994
,22, 4673–4680.
[CrossRef] [PubMed]
36.
Clement, M.; Posada, D.C.K.A.; Crandall, K.A. TCS: A computer program to estimate gene genealogies. Mol. Ecol.
2000
,9,
1657–1659. [CrossRef]
37.
Gallelli, A.; Galli, M.; De Simone, D.; Zaccardelli, M.; Loreti, S. Phenotypic and genetic variability of Pectobacterium carotovorum
isolated from artichoke in the Sele valley. J. Plant Pathol. 2009,91, 757–761.
38.
Alvarado, I.C.M.; Michereff, S.J.; Mariano, R.L.R.; Souza, E.B.; Quezado-Duval, A.M.; Resende, L.V.; Cardoso, E.; Mizubuti, E.S.G.
Characterization and Variability of Soft Rot-Causing Bacteria in Chinese Cabbage in North Eastern Brazil. J. Plant Pathol.
2011
,93,
173–181.
39.
Nabhan, S.; Wydra, K.; Linde, M.; Debener, T. The use of two complementary DNA assays, AFLP and MLSA, for epidemic and
phylogenetic studies of pectolytic enterobacterial strains with focus on the heterogeneous species Pectobacterium carotovorum.
Plant Pathol. 2012,61, 498–508. [CrossRef]
40.
Ma, X.; Stodghill, P.; Gao, M.; Perry, K.L.; Swingle, B. Identification of Pectobacterium versatile causing blackleg of potato in New
York State. Plant Dis. 2021,105, 2585–2594. [CrossRef]
Microorganisms 2023,11, 2122 13 of 13
41.
Park, K.T.; Hong, S.M.; Back, C.G.; Cho, Y.J.; Lee, S.Y.; Ten, L.N.; Jung, H.Y. First Report of Pectobacterium versatile as the Causal
Pathogen of Soft Rot in Kimchi Cabbage in Korea. Res. Plant Dis. 2023,29, 72–78. [CrossRef]
42.
Parvin, S.M.R.; Taghavi, S.M.; Osdaghi, E. Field surveys indicate taxonomically diverse Pectobacterium species inducing soft rot of
vegetables and annual crops in Iran. Plant Pathol. 2023,72, 1260–1271. [CrossRef]
43.
Oskiera, M.; Kału˙
zna, M.; Kowalska, B.; Smoli´nska, U. Pectobacterium carotovorum subsp. odoriferum on cabbage and Chinese
cabbage: Identification, characterization and taxonomic relatedness of bacterial soft rot causal agents. J. Plant Pathol.
2017
,1,
149–160.
44.
Arizala, D.; Arif, M. Genome-wide analyses revealed remarkable heterogeneity in pathogenicity determinants, antimicrobial
compounds, and CRISPR-cas systems of complex phytopathogenic genus Pectobacterium.Pathogens
2019
,8, 247. [CrossRef]
[PubMed]
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