Diversity of Bacterial Soft Rot-Causing Pectobacterium Species Affecting Cabbage in Serbia

The aim of this work was to identify and characterize the pectolytic bacteria responsible for the emergence of bacterial soft rot on two summer cabbage hybrids (Cheers F1 and Hippo F1) grown in the Futog locality (Bačka, Vojvodina), known for the five-century-long tradition of cabbage cultivation in Serbia. Symptoms manifesting as soft lesions on outer head leaves were observed during August 2021, while the inner tissues were macerated, featuring cream to black discoloration. As the affected tissue decomposed, it exuded a specific odor. Disease incidence ranged from 15% to 25%. A total of 67 isolates producing pits on crystal violet pectate (CVP) medium were characterized for their phenotypic and genotypic features. The pathogenicity was confirmed on cabbage heads. Findings yielded by the repetitive element palindromic-polymerase chain reaction (rep-PCR) technique confirmed interspecies diversity between cabbage isolates, as well as intraspecies genetic diversity within the P. carotovorum group of isolates. Based on multilocus sequence typing (MLST) using genes dnaX, mdh, icdA, and proA, five representative isolates were identified as Pectobacterium carotovorum (Cheers F1 and Hippo F1), while two were identified as Pectobacterium versatile (Hippo F1) and Pectobacterium odoriferum (Hippo F1), respectively, indicating the presence of diverse Pectobacterium species even in combined infection in the same field. Among the obtained isolates, P. carotovorum was the most prevalent species (62.69%), while P. versatile and P. odoriferum were less represented (contributing by 19.40% and 17.91%, respectively). Multilocus sequence analysis (MLSA) performed with concatenated sequences of four housekeeping genes (proA, dnaX, icdA, and mdh) and constructed a neighbor-joining phylogenetic tree enabled insight into the phylogenetic position of the Serbian cabbage Pectobacterium isolates. Bacterium P. odoriferum was found to be the most virulent species for cabbage, followed by P. versatile, while all three species had comparable virulence with respect to potato. The results obtained in this work provide a better understanding of the spreading routes and abundance of different Pectobacterium spp. in Serbia.


Introduction
Cabbage (Brassica oleracea var. capitata L.) is one of the world's most important vegetable crops, as its high yield and adaptability have facilitated worldwide distribution [1]. Owing to the abundance of health-promoting phytochemicals (glucosinolates, polyphenols, vitamins, and proteins), cabbage features in the traditional cuisine of many countries and has also been traditionally used for medicinal purposes [2]. According to the data provided by the Food and Agriculture Organization Corporate Statistical Database (FAOSTAT) for 2020, globally, about 2,414,288 ha were designated for cabbage cultivation [3]. aceae family, four housekeeping genes (i.e., dnaX, icdA, mdh, and proA) were utilized in the present study for typing the pectolytic isolates obtained from cabbage.
Repetitive element palindromic PCR (rep-PCR) is widely recognized as a useful technique for profiling different Pectobacterium species. In their study, Norman et al. successfully used rep-PCR with primers for BOX-, ERIC-, and REP-PCR for strain-level differentiation of P. carotovorum populations isolated from nursery retention ponds and large hypereutrophic lakes [24]. In addition to the aforementioned rep-PCR techniques (BOX-, ERIC-, and REP-PCR), for the first time, Maisuria and Nerurkar [25] also used GTG 5 -PCR, due to its highest discriminatory power, to differentiate P. carotovorum strains from soil and diseased fruits and vegetables. According to Zoledowska et al. [26], REP-PCR is the most useful tool for grouping P. parmentieri potato strains and is superior to BOX-and ERIC-PCR. Thus, as BOX-, ERIC-, REP-, and GTG 5 -PCR are among the most commonly used rep-PCR methods for DNA profiling and are proven to be sufficiently discriminative to reveal subtle differences even among strains belonging to the same species, all four methods were adopted in the present study.
Given the expanding genetic diversity of Pectobacterium spp. and the lack of recent data on the agents causing soft rot on cabbage in Serbia, the goal of this study was to contribute to their identification and characterization based on molecular and pathogenic features.

Sample Collection and Pathogen Isolation
In August 2021, bacterial soft rot symptoms were noted on the summer cabbage hybrids Cheers F1 (Takii Seed, Field I (geographic coordinates 45.2507100, 19.7247660)) and Hippo F1 (Sakata Seed, Field II (geographic coordinates 45.2567200, 19.7275470)) grown in two fields located in Futog (Bačka, Vojvodina) of 0.5 ha and 1 ha size, respectively. Soft rot symptoms on outer leaves, along with deep maceration of inner head leaves accompanied by black discoloration, were found on the inspected plants ( Figure 1). Due to decomposition, the affected tissue exuded a specific odor. Disease incidence in the visited fields was estimated at 15−25%.

Pathogenicity on cabbage
The pathogenicity of 67 isolates obtained in this study was evaluated using cabbage heads (unknown cultivar). Before inoculation, cabbage heads were washed under running tap water, uniformly sprayed with 70% ethanol, and dried at room temperature. Isolates used for inoculation were grown in nutrient broth (NB, HiMedia Laboratories) at 26 °C Prior to isolation, collected samples were washed under running tap water and dried on filter paper at room temperature. Isolations were performed on crystal violet pectate (CVP) media [40] from the small leaf sections (2-3 mm) that encompassed the transition zones between healthy and diseased tissue. CVP plates were incubated at 26 • C. All bacterial colonies forming characteristic cavities on the medium were selected and transferred onto nutrient agar (NA) [41] to obtain pure cultures. Isolates were long-term stored at −80 • C in lysogeny broth (LB) [42] supplemented with 30% (v/v) of sterile glycerol.

Pathogenicity on Cabbage
The pathogenicity of 67 isolates obtained in this study was evaluated using cabbage heads (unknown cultivar). Before inoculation, cabbage heads were washed under running tap water, uniformly sprayed with 70% ethanol, and dried at room temperature. Isolates used for inoculation were grown in nutrient broth (NB, HiMedia Laboratories) at 26 • C for 48 h while shaking and were adjusted to approximately 1 × 10 8 CFU mL −1 . Inoculations were performed by puncturing holes in cabbage heads and filling them with bacterial suspensions (~200 µL). The assays were performed in two sets of three independent replicates. Inoculated cabbage heads were placed in plastic boxes which were kept under room temperature (25 ± 1 • C) and high humidity (90−100%) conditions. Sterile distilled water (SDW) was used as a negative control, while the P. carotovorum strain Pcc10, previously isolated from cabbage in Bosnia and Herzegovina [8], served as a positive control treatment.
Cabbage heads were visually observed daily in order to monitor the occurrence of soft rot symptoms and the disease progress until complete decay. Emergence of soft lesions around the holes 24 h after the inoculation of cabbage heads with the suspension of tested isolates was considered a pectolytic-positive reaction.

DNA Extraction
Genomic DNA from the 67 cabbage isolates was extracted according to the hexadecyltrimethylammonium bromide (CTAB) procedure described previously by Popović et al. [43].

Preliminary Identification
All cabbage isolates were preliminarily identified using specific primers (F0145/E2477) designed based on the partial sequence of gene pmrA (response regulator) of P. carotovorum [44]. The sequences of the used primers are listed in Table 1. PCR amplifications were performed in a mixture (25 µL) consisting of Thermo Scientific DreamTaq PCR Master Mix (2×) (12.5 µL), nuclease-free water (Thermo Scientific TM , Waltham, MA, USA) (9.5 µL), 10 µM primers (forward/reverse) (1 µL each), and sample DNA (1 µL), according to the conditions proposed by Kettani-Halabi et al. [44]. Presence of a band in the searched position of 666 bp was checked on 1% agarose gel in relation to the positive control P. carotovorum strain Pcc10 and 200-10,000 bp SmartLadder MW-1700-10 (Eurogentec). Genetic diversity among the obtained 67 cabbage isolates was evaluated using the rep-PCR fingerprinting method with two oligonucleotide primer pairs [ERIC1R/ERIC2 (ERIC-PCR) and REP1R-I/ REP2-I (REP-PCR)] and two single oligonucleotide primers [BOXA1R (BOX-PCR) and GTG 5 (GTG 5 -PCR)] corresponding to the interspersed repetitive sequence elements. Primer sequences of the used primers are listed in Table 1. The PCR mixture (25 µL) comprised 12.5 µL of Thermo Scientific DreamTaq PCR Master Mix (2×), 9.5 µL of nuclease-free water (Thermo Scientific TM , Waltham, MA, USA), 1 µL of each (forward/reverse) primer (10 µM) and 1 µL of sample DNA. PCR amplifications for BOX-, ERIC-, and REP-PCR were performed under the conditions proposed by Louws et al. [45], while the methodology described by Versalovic et al. [46] was adopted for GTG 5 -PCR. After amplification, PCR products (5 µL) were mixed with DNA Gel Loading Dye (6X) (Thermo Scientific TM , Waltham, MA, USA) (2 µL) and were visualized on 2% agarose gel (FastGene ® ) strained with Midori Green Advance (Nippon Genetics Europe, Düren, Germany). PCR products were electrophoretically separated for 2.5 h (at 90 V and 300 mA) before being checked under a UV transilluminator. The obtained patterns were compared using PyElph 1.4 program and were subsequently used for the construction of the unweighted pair group method with arithmetic mean (UPGMA) phylogenetic tree. To easily compare the patterns obtained with each of the four used rep-PCR primers and to select all isolates that were potentially genetically different, each tree cluster, representing one DNA fingerprinting pattern, was provided with a number (DNA fingerprinting group). One isolate representing each combination of obtained DNA fingerprinting groups was randomly selected for further characterization (MLST and MLSA, and virulence assessment).

Multilocus Sequence Typing and Analysis (MLST/MLSA)
DNA of the seven selected representative cabbage isolates (Pc2321, Pc3821, Pc4821, Pc5421, Pv6321, Po7521, and Pc8321) was amplified with the primers produced based on the partial sequences of four housekeeping genes (dnaX (dnaX-F/dnaX-R), icdA (icdA400F/ icdA977R), mdh (mdh2/mdh4), and proA (proAF1/proAR1)), encoding DNA polymerase III subunit tau, isocitrate dehydrogenase, malate dehydrogenase, and gamma-glutamyl phosphate reductase, respectively. Primer sequences of the used primers are listed in Table 1. For all reactions, PCR mixtures were created as described in the previous subsection for rep-PCR. PCR amplifications were performed under the conditions described by Sławiak et al. [22] for the gene dnaX, while those reported by Ma et al. [16] were adopted for the genes icdA and proA, and the strategy employed by Moleleki et al. [17] was utilized for the gene mdh. Presence of bands in the searched positions was checked on 1% agarose gel in relation to 200-10,000 bp SmartLadder MW-1700-10 (Eurogentec). PCR products were purified using the Qiagen QIAquick PCR Purification Kit before being sent to the Eurofins Genomics' DNA sequencing service (Germany) for sequencing. The obtained sequences were manually checked for quality and were compared with the strains deposited in the National Center for Biotechnology Information (NCBI) database using the nucleotide BLAST (BLASTn) tool. All newly identified sequences were deposited to the NCBI database to obtain the accession numbers.

Virulence Assessment
The virulence potential of the seven representative cabbage isolates (Pc2321, Pc3821, Pc4821, Pc5421, Pv6321, Po7521, and Pc8321) was assessed using cabbage heads (unknown cv.) and potato tubers (cv. Arizona). Prior to inoculation, cabbage heads and potato tubers were sterilized as previously described (Section 2.2), and approximately 100 surface punctures (around 5 mm in depth) were made using a needle to allow bacteria to penetrate the tissue. Bacterial suspensions were prepared as described for the pathogenicity test (Section 2.2) and SDW was used as a negative control. Inoculations were performed by spraying cabbage heads/potato tubers as uniformly as possible, making sure to cover the entire outer surface. The experiment was conducted in three independent replicates, with three cabbage heads (nine in total per isolate) and five potato tubers (15 in total per isolate) for each replicate. Two experiments-for disease progress rating five and seven days after inoculation (d.a.i.), respectively-were performed in parallel. For both experiments, the initial weights of cabbage heads and potato tubers were recorded. To obtain statistically comparable results, sums of the initial weights for each of the tested cabbage isolates were almost equal. The inoculated cabbage heads and potato tubers were placed in plastic boxes under high humidity conditions (90−100%). The experiment was performed during summer when the room temperature was 28 ± 2 • C.
Cabbage heads and potato tubers were weighed, whereby the mean values of the initial and the final (5 and 7 d.a.i.) weights were used to calculate the disease progress curve (AUDPC) according to the following equation: where y i = disease progress assessment at the ith observation, t i = time (days) at the ith observation, and n = total number of observations. All obtained values were statistically processed using Minitab 21 Statistical Software, whereby one-way analysis of variance (one-way ANOVA) was performed, and the resulting values were compared using Tukey's honestly significant difference (HSD) test. Values having p < 0.05 were considered statistically significant.

Isolation, Preliminary Identification, and Pathogenicity
In this study, 67 isolates were selected from cabbage hybrids Cheers F1 (34 isolates) and Hippo F1 (33 isolates), forming characteristic cavities on CVP medium and small, whitish, irregularly shaped colonies on NA (Table 3). All isolates produced bands at 666 bp after amplification with the Pectobacterium-specific primer pair F0145/E2477 (Table 3).  I  I  I  I  Pc2121  Cheers F1  +  +  I  I  I  I  Pc2221  Cheers F1  +  +  I  I  I  I  Pc2321  Cheers F1  +  +  I  I  I  I  Pc2421  Cheers F1  +  +  I  I  I  I  Pc2521  Cheers F1  +  +  I  I  I  I  Pc2621  Cheers F1  +  +  I  I  I  I  Pc2721  Cheers F1  +  +  I  I  I  I  Pc2821  Cheers F1  +  +  I  I  I  I  Pc3121  Cheers F1  +  +  II  II  II  II  Pc3221  Cheers F1  +  +  II  II  II  II  Pc3321  Cheers F1  +  +  II  II  II  II  Pc3421  Cheers F1  +  +  II  II  II  II  Pc3521  Cheers F1  +  +  II  II  II  II  Pc3621  Cheers F1  +  +  II  II  II  II  Pc3721  Cheers F1  +  +  II  II  II  II  Pc3821  Cheers F1  +  +  II  II  II  II  Pc3921  Cheers F1  +  +  II  II  II  II  Pc4221  Cheers F1  +  +  III  II  III  III  Pc4321  Cheers F1  +  +  III  II  III  III  Pc4421  Cheers F1  +  +  III  II  III  III  Pc4521 Cheers F1 + + III II III III  +  +  III  II  III  III  Pc4721  Cheers F1  +  +  III  II  III  III  Pc4821  Cheers F1  +  +  III  II  III  III  Pc4921  Cheers F1  +  +  III  II  III  III  Pc5021  Cheers F1  +  +  IV  III  IV  III  Pc5121  Cheers F1  +  +  IV  III  IV  III  Pc5221 Cheers Hippo F1 Hippo The pathogenicity of all cabbage isolates was confirmed on cabbage heads by visually identifying irregularly shaped soft lesions (approximately 3−5 cm in diameter) around the inoculation points (holes) 1 d.a.i. The diameter of decomposing tissue enlarged daily, and the affected area spread from the outer leaves to the inner tissues, while causing tissue discoloration from cream to black. At 5 d.a.i., cabbage heads were almost completely macerated and exuded a specific odor.

Rep-PCR
The UPGMA trees showing genetic diversity among the 67 tested cabbage isolatesconstructed based on the obtained BOX-, ERIC-, GTG 5 -, and REP-PCR banding patternsare shown in Figure S1, which also features virtual gel images of rep-PCR patterns corresponding to each group of isolates. Based on the differences found, each tree cluster was assigned a different number: BOX (I-VII), ERIC (I-VI), GTG 5 (I-VII), and REP (I-VI) ( Table 3). The obtained results indicate that the tested isolates are genetically diverse. BOXand GTG 5 -PCR generated seven (I-VII), while ERIC-and REP-PCR generated six distinct banding patterns (I-VI). Based on the BOX-and GTG 5 -PCR findings, isolates were divided into the same seven groups, namely I: Pc2021-Pc2821, II: Pc3121-Pc3921, III: Pc4221-Pc4921, IV: Pc5021-Pc5721, V: Pv1021-Pv1621 and Pv6121-Pv6621, VI: Po7021-Po7521 and Po9121-Po9621, and VII: Pc8021-Pc8721 (Table 3). However, ERIC-and REP-PCR did not separate the isolates into six identical groups. ERIC-PCR implied the homogeneity of isolates Pc3121-Pc3921 and Pc4221-Pc4921 by placing them in the same tree cluster (DNA fingerprinting group II), while REP-PCR indicated homogeneity of isolates Pc4221-Pc4921 and Pc5021-Pc5721 (DNA fingerprinting group III) ( Table 3). The distribution of the remaining isolates within the UPGMA groups remained the same and coincided with that obtained for BOX-and GTG 5 -PCR. Based on the combined results obtained with all four primers, seven isolates (Pc2321, Pc3821, Pc4821, Pc5421, Pv6321, Po7521, and Pc8321), each representing one group on the UPGMA tree (i.e., one DNA banding pattern), were randomly selected for further characterization.
The NJ phylogenetic tree generated based on the concatenated sequences of genes dnaX, icdA, mdh, and proA is presented in Figure 2. Based on these genes, the tested and comparative P. carotovorum, P. versatile, and P. odoriferum isolates/strains were separated into three clusters within the tree, each corresponding to one species. However, genetic heterogeneity (i.e., intraspecies genetic diversity) was observed within each species. Five of the seven tested cabbage P. carotovorum isolates examined in this study were separated into four groups (I: Pc2321 and Pc4821, II: Pc3821, III: Pc5421, and IV: Pc8321) within the cluster. The remaining tested P. versatile isolate Pv6321 was the most closely related to the comparative P. versatile strain DSM 30169 isolated from cabbage in Germany, while the P. odoriferum isolate Po7521 was most similar to the type P. odoriferum strain CFBP 1878 isolated from chicory in France. D. solani strain RNS 05.1.2A was placed on a monophyletic tree branch as an outgroup.

Virulence assessment
The developed disease symptoms observed seven days after the spray-inoculation of cabbage heads and potato tubers with suspensions of the tested isolates are presented in Figure 3. The neighbor-joining phylogenetic tree constructed based on the concatenated sequences of genes dnaX, icdA, mdh, and proA for seven representative P. carotovorum , P. odoriferum , and P. versatile isolates examined in this study and 19 strains of P. carotovorum, P. odoriferum, and P. versatile isolated from various hosts and countries, which were retrieved from the GenBank. The tree was rooted with the D. solani strain RNS 05.1.2A.

Virulence assessment
The developed disease symptoms observed seven days after the spray-inoculation of cabbage heads and potato tubers with suspensions of the tested isolates are presented in Figure 3. The neighbor-joining phylogenetic tree constructed based on the concatenated sequences of genes dnaX, icdA, mdh, and proA for seven representative P. carotovorum , P. odoriferum , and P. versatile isolates examined in this study and 19 strains of P. carotovorum, P. odoriferum, and P. versatile isolated from various hosts and countries, which were retrieved from the GenBank. The tree was rooted with the D. solani strain RNS 05.1.2A.

Virulence assessment
The developed disease symptoms observed seven days after the spray-inoculation of cabbage heads and potato tubers with suspensions of the tested isolates are presented in Figure 3. The neighbor-joining phylogenetic tree constructed based on the concatenated sequences of genes dnaX, icdA, mdh, and proA for seven representative P. carotovorum , P. odoriferum , and P. versatile isolates examined in this study and 19 strains of P. carotovorum, P. odoriferum, and P. versatile isolated from various hosts and countries, which were retrieved from the GenBank. The tree was rooted with the D. solani strain RNS 05.1.2A.

Virulence assessment
The developed disease symptoms observed seven days after the spray-inoculation of cabbage heads and potato tubers with suspensions of the tested isolates are presented in Figure 3.
isolates examined in this study and 19 strains of P. carotovorum, P. odoriferum, and P. versatile isolated from various hosts and countries, which were retrieved from the GenBank. The tree was rooted with the D. solani strain RNS 05.1.2A.

Virulence Assessment
The developed disease symptoms observed seven days after the spray-inoculation of cabbage heads and potato tubers with suspensions of the tested isolates are presented in Figure 3. Figure 2. The neighbor-joining phylogenetic tree constructed based on the concatenated sequences of genes dnaX, icdA, mdh, and proA for seven representative P. carotovorum , P. odoriferum , and P. versatile isolates examined in this study and 19 strains of P. carotovorum, P. odoriferum, and P. versatile isolated from various hosts and countries, which were retrieved from the GenBank. The tree was rooted with the D. solani strain RNS 05.1.2A.

Virulence assessment
The developed disease symptoms observed seven days after the spray-inoculation of cabbage heads and potato tubers with suspensions of the tested isolates are presented in Figure 3. The results of the AUDPC analysis of the tested isolates on cabbage heads and potato tubers are shown in Figure 4A,B, respectively.
The AUDPC values pertaining to cabbage heads ranged from 4964.2 to 5990.46 for the P. odoriferum isolate Po7521 and P. carotovorum isolate Pc8321, respectively ( Figure 4A). Based on the obtained AUDPC values, the P. odoriferum isolate Po7521 exhibited the highest virulence potential, followed by the P. versatile isolate Pv6321, while the P. carotovorum isolates Pc2321, Pc3821, Pc4821, Pc5421, and Pc8321 exhibited the lowest (and comparable) virulence potential. Statistically significant differences between the initial (0 d.a.i.) and the final (5 and 7 d.a.i.) weights were observed after cabbage inoculation with the P. carotovorum isolates Pc2321 and Pc3821, as well as the P. odoriferum isolate Po7521. On the other hand, the weights measured 5 and 7 d.a.i. with the P. versatile isolate Pv6321 were comparable but differed significantly from the initial weight (0 d.a.i.).
The AUDPC values pertaining to potato tubers ranged from 249.92 to 342.15 for the P. carotovorum isolates Pc2321 and Pc4821, respectively ( Figure 4B). As the AUDPC values obtained for the seven representative isolates were comparable, all tested isolates appeared to be equally virulent with respect to this host. However, the weights measured 5 and 7 d.a.i. were statistically significantly lower than the initial weights when samples were inoculated with the P. carotovorum isolates Pc4821, Pc5421, and Pc8321. For the remaining two P. carotovorum isolates (Pc2321 and Pc3821), one P. versatile Pv6321, and one P. odoriferum Po7521, the weights measured 5 and 7 d.a.i. were comparable, but differed significantly from the initial values (0 d.a.i.). Figure 3. Examples of the bacterial soft rot symptoms on the cabbage head and potato tuber that were observed seven days after inoculation with the Serbian P. versatile strain Pv6321 and the P. carotovorum strain Pc3821, respectively.
The results of the AUDPC analysis of the tested isolates on cabbage heads and potato tubers are shown in Figure 4A,B, respectively. The AUDPC values pertaining to cabbage heads ranged from 4964.2 to 5990.46 for the P. odoriferum isolate Po7521 and P. carotovorum isolate Pc8321, respectively ( Figure 4A). Based on the obtained AUDPC values, the P. odoriferum isolate Po7521 exhibited the highest virulence potential, followed by the P. versatile isolate Pv6321, while the P. carotovorum isolates Pc2321, Pc3821, Pc4821, Pc5421, and Pc8321 exhibited the lowest (and comparable) virulence potential. Statistically significant differences between the initial (0 d.a.i.) and the final (5 and 7 d.a.i.) weights were observed after cabbage inoculation with the P. carotovorum isolates Pc2321 and Pc3821, as well as the P. odoriferum isolate Po7521. On the other hand, the weights measured 5 and 7 d.a.i. with the P. versatile isolate Pv6321 were comparable but differed significantly from the initial weight (0 d.a.i.).

Discussion
To the best of our knowledge, this is the first study since the pioneering work of Arsenijević and Obradović published more than 20 years ago [47], in which Erwinia carotovora subsp. carotovora was identified in the Bačka region, to provide evidence on the presence and diversity of the three bacteria (i.e., P. carotovorum, P. odoriferum, and P. versatile) causing soft rot in cabbage in Vojvodina (Serbia). The obtained results indicated presence of a combined infection in Field II, where all three identified species were confirmed on the cabbage hybrid Hippo F1. However, in Field I (hybrid Cheers F1), only P. carotovorum was detected. Analysis of the 67 cabbage isolates indicated that P. carotovorum was the most represented (62.69%), followed by P. versatile (19.40%) and P. odoriferum (17.91%). These findings are not surprising, given that P. carotovorum species is generally recognized as the main causal agent of soft rot in Brassicaceae plants [28]. This species was described on cabbage and Chinese cabbage in China, Brazil, Malaysia, Korea, and Bosnia and Herzegovina [8,[48][49][50][51]. However, there is paucity of data on the presence of P. versatile and P. odoriferum on cabbage. In addition to P. carotovorum, P. versatile, and P. odoriferum, Chen et al. [31] also reported species P. aroidearum, P. brasiliense, and P. polaris on Chinese cabbage grown in different districts of Beijing (China). Presence of P. odoriferum was also reported on cabbage and Chinese cabbage in Central Poland, China, and Iran [28,48,52]. However, the lack of data on the presence of P. odoriferum and P. versatile on cabbage does not imply its absence on this host, as it is at least partly due to their recent reclassification (i.e., elevation from subspecies to the species level) within the genus Pectobacterium [53].
It is known that the genus Pectobacterium includes heterogonous strains characterized by diverse biochemical, physiological, and genetic properties even within the same species [54]. Based on the rep-PCR results, the Pectobacterium spp. isolates tested as a part of the present study were genetically heterogeneous, forming seven (BOX-and GTG 5 -PCR) and six (ERIC-and REP-PCR) groups on the UPGMA tree, depending on the utilized primers. In other words, the rep-PCR results indicate interspecies genetic heterogeneity, as well as intraspecies heterogeneity within P. carotovorum isolates only, which were clustered in five (BOX-and GTG 5 -PCR) and four (ERIC-and REP-PCR) groups. The rep-PCR (BOX-, ERIC-, and REP-PCR) analysis conducted by Alvarado et al. [49] also revealed high genetic variability among P. carotovorum strains isolated from Chinese cabbage in Pernambuco state (north-east Brazil). Based on the rep-PCR with primers for BOX-, ERIC-, and REP-PCR, Loc et al. [38] reached a similar conclusion to the one put forward in this study, positing that Pectobacterium strains of the same species tend to group closely according to their respective taxonomic designations. Considering that authors of several extant studies singled out rep-PCR as discriminative enough to reveal subtle differences between different Pectobacterium spp., as well as those among the same species, as proposed by Zoledowska et al. [26] for P. parmentieri strains from Poland, rep-PCR is a promising technique for the clarification of genetic diversity and discrimination of soft rot-causing Pectobacterium spp.
In the present study, typing of four housekeeping genes (dnaX, icdA, mdh, and proA) enabled appropriate identification of the tested cabbage isolates and a clear separation of each of the three identified species from one another. The existence of inter-and intraspecies genetic heterogeneity between the three detected Pectobacterium species was again confirmed based on MLSA with concatenated sequences of the same four genes. According to Zeigler [55], among genes present in all sequenced bacterial genomes, the dnaX gene is considered one of the best candidates for assigning bacterial strains to the species level. Sławiak et al. [22] also highlighted the usefulness of gene dnaX for the identification of European potato Dickeya spp. strains. However, despite the demonstrated high resolution of the dnaX gene, the use of other protein-coding genes should not be discouraged due to the well-known claim that mutations, as the main engine of evolution, in Pectobacterium spp. mostly occur on these genes [14]. Moreover, using multiple genes allows for a greater genome coverage, which undoubtedly leads to a more reliable phylogeny reconstruction. In extant research, the remaining three genes (icdA, mdh, and proA) were successfully used for the typing of Pectobacterium spp. in different combinations with other housekeeping genes (e.g., acnA, gapA, mtlD, pgi, recA, rpoS, etc.) [16][17][18][19][20]. Important parameters when selecting these genes are (i) ubiquity in most enterobacteria, (ii) high discriminatory ability, and (iii) indispensable role in key metabolic processes [16].
In the virulence assessment assay performed as a part of this work, the lowest AUDPC values pertaining to cabbage heads (i.e., the highest virulence potential) was observed for the P. odoriferum isolate Po7521, followed by the P. versatile isolate Pv6321. On the other hand, the P. carotovorum isolates Pc2321, Pc3821, Pc4821, Pc5421, and Pc8321 were the least virulent, with a comparable virulence potential for cabbage. However, such statistical differences in aggressiveness among isolates/species were not detected on potato tubers. While these findings may be indicative of host−pathogen specificity in the case of bacteria P. odoriferum and P. versatile, more confident claims about such interactions would require more extensive studies performed on a larger number of known hosts. Bearing in mind the wide distribution, ubiquity, and polyphagous nature of P. carotovorum [56], and the resulting genetic diversity that enabled this species to survive and adapt to different ecological niches (i.e., hosts), it is likely that differences in aggressiveness will be observed between different hosts. In the study conducted by Li et al. [57], P. odoriferum strains isolated from Chinese cabbage exhibited much higher virulence potential for Chinese cabbage compared to the tested P. carotovorum and P. brasiliensis strains (measured 24 h and 30 h post-inoculation), all obtained from the same host. These authors did not observe statistically significant differences in the virulence potential between P. carotovorum and P. brasiliensis strains. However, it remains to be established whether the differences between species would be sustained for the duration of the disease progression. Moreover, based on the results reported by Waleron et al. [58], no statistically significant differences in the virulence potential on potato were observed between P. carotovorum and P. odoriferum, or between P. carotovorum and P. brasiliensis, based on the analyses performed 3 d.a.i. This research significantly contributes to the current knowledge of the diversity of pectolytic bacteria affecting cabbage in Serbia, which has so far remained unexplored despite their great importance.