The International Trade of Ware Vegetables and Orna-Mental Plants—An Underestimated Risk of Accelerated Spreading of Phytopathogenic Bacteria in the Era of Globalisation and Ongoing Climatic Changes

Bacteria of the genus Pectobacterium are globally occurring pathogens that infect a broad spectrum of plants. The plant cell wall degrading enzymes allow them to cause diseases like soft rot and blackleg. Worldwide trade and exchange of plant material together with the accompanying microorganisms contributed to the rapid spread and consequently the acquisition of new traits by bacteria. The 161 pectinolytic strains were isolated from symptomless vegetables and ornamental plants acquired from Polish and foreign local food markets. All strains except four Dickeya isolates were identified as belonging to the Pectobacterium genus by PCR with species-specific primers and recA gene sequencing. The newly isolated bacteria were assigned to eight species, P. versatile (50 strains), P. carotovorum (33), P. brasiliense (27), P. atrosepticum (19), P. parmentieri (12), P. polaris (11), P. parvum (3) and P. odoriferum (2). ERIC PCR and phenotypic characteristics revealed high heterogeneity among P. carotovorum, P. brasiliense and P. versatile isolates. Moreover, a subset of the newly isolated strains was characterised by high tolerance to changing environmental conditions such as salinity, pH and water availability. These bacteria can effectively macerate the tissues of various plants, including potato, chicory and orchid. Our results indicate that Pectobacterium strains isolated from internationally traded, symptomless vegetables and ornamental plants have high potential for adaptation to adverse environmental conditions and to infect various host plants. These features may contribute to the success of the genus Pectobacterium in spreading between different climatic zones and facilitate the colonisation of different ecological niches.


Introduction
Bacteria of the genus Pectobacterium are widespread and cause many diseases on a wide range of economically important plants worldwide. Those bacteria have a broad host range, and most do not demonstrate specificity for host plants. Although the genus Pectobacterium is known mainly as plant pathogens, they may also be saprophytes [1]. Nevertheless, the infections with these pathogens lead to severe economic losses of horticultural and ornamental plants in the fields and during their storage. Thus, the genus is recognised as one of the ten most important bacterial plant pathogens and is the subject of research by numerous groups of scientists [2]. Furthermore, among 157 strains identified as Pectobacterium, nineteen strains were classified as P. atrosepticum, twelve strains isolated from symptomless potatoes as P. parmentieri, and ten strains were identified as P. brasiliense, based on species-specific PCR reactions [29][30][31]. Unfortunately, 115 out of 157 newly isolated strains could not be classified to the species level with applied species-specific primers ( Table 1).
The genetic diversity of 161 new Pectobacterium and Dickeya isolates originating from symptomless plants and 31 reference strains isolated from symptomatic plants was assessed by the ERIC-PCR method. In total, 23 different ERIC profiles were discriminated (Table 1).
In the next step, based on obtained results, 75 strains with different ERIC fingerprints and different geographical origins or isolated from distinct plant materials were selected for the recA gene sequencing. Finally, the obtained sequences of newly isolated strains were compared with the recA gene sequences available in the Genbank and the phylogenetic analysis was conducted.
It should be noted that different Pectobacterium species can occur on the same asy tomatic plant sample. For example, the presence of two species, P. brasiliense and P. pol has been detected on Sugar beet sample 111 (DPMP394 and DPMP403), Bittersweet s ple 112 (DPMP396 and DPMP397) and Moroccan potato tuber sample 13 (DPMP55 DPMP730). On the other hand, from the same potato tuber samples, the other comb tions of Pectobacterium species have been isolated. P. atrosepticum and P. versatile were tected in Potato stem sample 98 from Poland (DPMP344 and DPMP133) and Potato tu from Belgium (DPMP133 and DPMP134). P. atrosepticum DPMP278 was co-isolated w P. polaris DPMP280 from Potato tuber sample 73 from Norway. Meanwhile, in Poland P. versatile DPMP352 strain was co-isolated with the P. parmentieri DPMP353 strain f the same sample of potato tuber 99.1. Another two Pectobacterium species combinati P. parmentieri DPMP370 and P. carotovorum DPMP369 and P. parvum DPMP20 and P. satile MKW18 were extracted from Cyprus potato tuber samples number 100 and mkw respectively. Furthermore, three different species: P. brasilense (strain DPMP152), P. ca vorum (strain DPMP146), and P. versatile (strain DPMP155) were observed on the s potato tuber sample number 44 from Morocco. Meanwhile, from Potato tuber sam number 155 from Kazakhstan, P. atrosepticum DPMP634, P. carotovorum DPMP331 an versatile DPMP632 and DPMP633 were detected (Table 2).  The most abundant species were P. versatile (30%), P. carotovorum (20%), P. brasiliense (16%), and P. atrosepticum (11%) that have been isolated from different plant species (vegetables and ornamental plants as well), originating from different countries. Only from Potatoes have we isolated all detected Pectobacterium species (Table 1).
It should be noted that different Pectobacterium species can occur on the same asymptomatic plant sample. For example, the presence of two species, P. brasiliense and P. polaris, has been detected on Sugar beet sample 111 (DPMP394 and DPMP403), Bittersweet sample 112 (DPMP396 and DPMP397) and Moroccan potato tuber sample 13 (DPMP55 and DPMP730). On the other hand, from the same potato tuber samples, the other combinations of Pectobacterium species have been isolated. P. atrosepticum and P. versatile were detected in Potato stem sample 98 from Poland (DPMP344 and DPMP133) and Potato tuber from Belgium (DPMP133 and DPMP134). P. atrosepticum DPMP278 was co-isolated with P. polaris DPMP280 from Potato tuber sample 73 from Norway. Meanwhile, in Poland, the P. versatile DPMP352 strain was co-isolated with the P. parmentieri DPMP353 strain from the same sample of potato tuber 99.1. Another two Pectobacterium species combinations, P. parmentieri DPMP370 and P. carotovorum DPMP369 and P. parvum DPMP20 and P. versatile MKW18 were extracted from Cyprus potato tuber samples number 100 and mkw18, respectively. Furthermore, three different species: P. brasilense (strain DPMP152), P. carotovorum (strain DPMP146), and P. versatile (strain DPMP155) were observed on the same potato tuber sample number 44 from Morocco. Meanwhile, from Potato tuber sample number 155 from Kazakhstan, P. atrosepticum DPMP634, P. carotovorum DPMP331 and P. versatile DPMP632 and DPMP633 were detected ( Table 2). The ability to macerate plant tissue was investigated for randomly selected strains representing each of the isolated eight Pectobacterium and one Dickeya species. The number of strains selected for adaptation tests reflects the percentage of strains classified into each Pectobacterium species detected on asymptomatic plants. The pathogenicity of 47 strains was assessed by maceration of potato tuber, while, for 44 strains, the assay was performed on chicory and iris leaves. None of the selected strains caused disease on Iris leaves (data not shown). All the tested strains were capable to macerate potato tuber tissues; among them, 41 strains caused soft rot of potato tubers and chicory leaves also. However, five strains: P. atrosepticum DPMP634, P. brasiliense IFB5258, P. parmentieri DPMP353 and SCC3193, as well as P. versatile DPMP633 macerated efficiently potato tubers but exhibited weak pathogenicity on chicory. Other four strains, P. atrosepticum ICMP1526 T , P. carotovorum LMG2404 T , P. parmentieri IFB5322, and D. dadantii DPMP625, caused maceration of potato tuber tissue and did not cause significant damage to chicory leaves (Figures 3 and 4, Tables S1 and S2).
Of 47 strains for which the potato tuber maceration capacity was tested, 27 were isolated in this study from asymptomatic plant samples, and the remaining 20 (marked with asterisks in Table S1 and Figures 3 and 4) were the reference strains or originated from plants with disease symptoms. Both groups of strains were characterised by the ability to macerate both potato tubers and chicory leaves.   Of 47 strains for which the potato tuber maceration capacity was tested, 27 were isolated in this study from asymptomatic plant samples, and the remaining 20 (marked with  Strains isolated from symptomless plants have nearly similar ability to macerate plant tissues as strains retrieved from plants with disease symptoms (Figures 3 and 4, Figures S1 and S2, Tables S1 and S2). However, for the strains originated from symptomatic plants, a slightly smaller rot area was observed than for asymptomatic strains ( Figure 5, Table S2). The p-value was 0.02 according to the ANOVA with Welch corrections for nonhomogeneous variances criterion followed by a post-hoc Games-Howell analysis ( Figure 5).
Pathogens 2022, 11, x FOR PEER REVIEW 13 of 27 asterisks in Table S1 and Figures 3 and 4) were the reference strains or originated from plants with disease symptoms. Both groups of strains were characterised by the ability to macerate both potato tubers and chicory leaves. Strains isolated from symptomless plants have nearly similar ability to macerate plant tissues as strains retrieved from plants with disease symptoms (Figures 3 and 4, Figure S1 and S2, Tables S1 and S2). However, for the strains originated from symptomatic plants, a slightly smaller rot area was observed than for asymptomatic strains ( Figure 5, Table S2). The p-value was 0.02 according to the ANOVA with Welch corrections for nonhomogeneous variances criterion followed by a post-hoc Games-Howell analysis ( Figure  5).

Adaptation for Various Environmental Conditions
Adaptation tests for different environmental conditions: variable pH, salinity levels and water availability were performed for 35 Pectobacterium strains (Tables 3-5). Strains representing each of identified Pectobacterium species were randomly selected.
pH Influence on Bacterial Growth All tested strains showed the ability to grow in a Tryptic Soy Broth (TSB) medium with a pH of 5 to 10 but their growth efficiency declined with the more alkaline pH. Only five strains, one strain P. brasiliense DPMP55, isolated from a Moroccan potato, and two strains of P. carotovorum DPMP199 and DPMP200, also isolated from potatoes originating in Egypt, as well as P. polaris DPMP286 and P. versatile DPMP198 were able to tolerate low pH (pH= 4). On the contrary, the highest pH value = 11 was tolerated by all strains except two P. brasiliense strains, DPMP68 from symptomatic potato and DPMP396 from Bittersweet, and one P. polaris DPMP403 from Sugar beet grown in Poland. All tested strains showed their optimum growth at a slightly acidic pH of 5 or 6, except that P. brasiliense strain DPMP396 and P. parvum DPMP20, which showed their optimal at a neutral pH = 7. Only three strains, P. brasiliense DPMP55, P. polaris DPMP286, and P. versatile DPMP198 were able to grow and were metabolically active in all tested pH ranges, from 4 up to 11 (Table 3).

Adaptation for Various Environmental Conditions
Adaptation tests for different environmental conditions: variable pH, salinity levels and water availability were performed for 35 Pectobacterium strains (Tables 3-5). Strains representing each of identified Pectobacterium species were randomly selected.
pH Influence on Bacterial Growth All tested strains showed the ability to grow in a Tryptic Soy Broth (TSB) medium with a pH of 5 to 10 but their growth efficiency declined with the more alkaline pH. Only five strains, one strain P. brasiliense DPMP55, isolated from a Moroccan potato, and two strains of P. carotovorum DPMP199 and DPMP200, also isolated from potatoes originating in Egypt, as well as P. polaris DPMP286 and P. versatile DPMP198 were able to tolerate low pH (pH= 4). On the contrary, the highest pH value = 11 was tolerated by all strains except two P. brasiliense strains, DPMP68 from symptomatic potato and DPMP396 from Bittersweet, and one P. polaris DPMP403 from Sugar beet grown in Poland. All tested strains showed their optimum growth at a slightly acidic pH of 5 or 6, except that P. brasiliense strain DPMP396 and P. parvum DPMP20, which showed their optimal at a neutral pH = 7. Only three strains, P. brasiliense DPMP55, P. polaris DPMP286, and P. versatile DPMP198 were able to grow and were metabolically active in all tested pH ranges, from 4 up to 11 ( Table 3).

The Salinity Impact on Bacterial Growth
Most of the tested strains (31 out of 35) showed their optimum growth at very low salinity up to 1% of NaCl in the medium. Only two strains, P. brasiliense DPMP396, and P. carotovorum DPMP199 grew best in medium containing 4 and 3% NaCl, respectively. Three P. parmentieri strains DPMP390, IFB5322, and SCC3193 were the most salinity-sensitive and only grew in a medium containing less than 4% NaCl. A vast spectrum of salinity from 0 to 8% NaCl in the medium was tolerated by the strain P. brasiliense DPMP55. In salinity equal to 11%, none of the tested isolates maintained an active metabolism, as evidenced by the resazurin test. However, ten strains, P. carotovorum DPMP199 and DPMP399, P. brasiliense DPMP55, DPMP224, DPMP372, DPMP396, and DPMP374, P. atrosepticum DPMP275, P. versatile DPMP402 and DPMP452 can survive in medium containing 11% of NaCl and were viable in a spot test performed on an MH medium without NaCl (Table 4).
The symbol "X" means no bacterial growth in the spot test, "V" means the presence of viable bacterial cells in spot test. The "OPT" symbol indicates the optimum at which the strain reached the highest OD value. * reference strains and strains that were isolated from plants with disease symptoms. T -Type Strain. The following colours indicate average OD ranges of two measurements. Black: <0.03, grey: 0.03-0.3, blue: 0.3-0.7, white: >0.7.

The Impact of Variable Water Availability on Bacterial Growth
All tested strains achieved the highest optical density value in medium without the addition of PEG. Out of 35 tested isolates, 13 were viable at PEG concentration above 200 g L −1 . Four Pectobacterium strains were viable in the TSB medium containing 300-400 g L −1 PEG, and two P. versatile strains DPMP198 and DPMP202 were isolated from cactus and P. carotovorum strains DPMP199 and DPMP200 that were isolated from potato that was grown in Egypt. The most resistant was one P. carotovorum strain DPMP202 isolated from cactus, which remained viable and active metabolically at a concentration of PEG equal to 500 g L −1 (Table 5). Table 4. Growth in various salinity conditions.

Concentration of Sodium Chloride [%]
The symbol "X" means no bacterial growth in the spot test, "V" means the presence of viable bacterial cells in a spot test. The "OPT" symbol indicates the optimum at which the strain reached the highest OD value. * reference strains and strains that were isolated from plants with disease symptoms. T -Type Strain. The average OD ranges are highlighted in the same color as in Table 3. Table 5. The tolerance for limited water availability.

Concentration Polyethylene Glycol [g/L] Strain
0 g/L 50 g/L 75 g/L 100 g/L 200 g/L 300 g/L 400 g/L 500 g/L Among 34 tested isolates, 13 strains, were resistant to ampicillin, 9 strains to erythromycin, 2 to gentamicin, 6 strains to kanamycin, and 5 to streptomycin and tetracycline (Table 6). The most susceptible to tested antibiotics were P. atrosepticum, P. brasiliense and P. polaris strains. The latter one revealed sensitivity to all of tested antibiotics. In contrast, P. versatile strains were most resistant among tested Pectobacterium species. Seven out of 9 P. versatile strains were resistant to ampicillin, 5 to the erythromycin, 4 to kanamycin and tetracycline, 3 to streptomycin, and 2 were growing in the presence of gentamicin.
We additionally observed that some strains of P. brasiliense, P. carotovorum and P versatile were able to grow on ESBL, while 3 P. versatile, 1 P. odoriferum and 1 P. parmentieri strains grew on CARBA chromagar plates (Table 6).
Two P. versatile strains DPMP198 and DPMP202, which were isolated from cactus and strain DPMP633 from Kazakhstan, were resistant to all tested antibiotics and revealed an ESBL type of resistance. Three other P. versatile strains DPMP108 and DPMP112 isolated from Iris and DPMP181 from zucchini in Poland exhibited resistance to carbapenems.

Discussion
In the period of 1999-2018, we collected 241 samples of asymptomatic ware vegetable, ornamental, and herbaceous plant samples. From 149 (62%) of symptomless plants that came from twenty-two countries from Africa, America, Asia, and Europe samples, we have isolated 161 pectinolytic bacteria.
Genetic identification revealed that 98% of isolates belonged to eight Pectobacterium spp., and only 2% of isolates were classified as Dickeya. However, it should be noted that bacteria were isolated at temperature 28 • C, which is not optimal for Dickeya growth.
Among Pectobacterium species, P. versatile was most frequently isolated from symptomless plants. Likewise, it was the most abundant Pectobacterium species deposited in CIRM-CFBP-a French collection of plant pathogenic bacteria [36]. Furthermore, this observation agrees with our earlier studies from 2001 and 2002 [37,38]. Analysis of the recA PCR-RFLP profiles for strains present in Polish and international collections of Erwinia carotovora indicated that only seven profiles: 3, 4, 5, 6, 7, 13 and 18 were common for both groups. Four profiles: 3, 4, 5, 6 were predominant in Polish and worldwide collection, and, in both populations, about 44% of the collected strains belonged to profile number 4. Currently, profiles 4 and 5 gather P. versatile strains, while profiles 3 and 6 are characteristic for P. parmentieri and P. polaris species, respectively. The sequencing of the recA gene for strains that have been used for RFLP analysis of an amplified fragment of recA gene confirmed that the most frequently observed RFLP profiles 4 and 5 are typical for P. versatile species. Furthermore, P. versatile was also most frequently isolated from water [39]. Thus, it is possible to conclude that, for the last twenty years, P. versatile continues to be the most frequently isolated taxon among the Pectobacterium species.
Strains belonging into P. versatile together with P. brasiliense, P. carotovorum, and P. polaris are the most divergent among Pectobacterium species. For each species, four different fingerprinting profiles were observed. In the case of P. atrosepticum and P. parmentieri, three and two ERIC profiles were determined, respectively. The above observations are in line with numerous reports on genetic diversity within the species belonging to the genus Pectobacterium. It should be noted that strains isolated from symptomatic plants do not differ genetically from those isolated from asymptomatic plants. Both groups of strains are assigned to the same fingerprinting profiles and recA gene sequences.
It should be emphasised that, in this study, we have detected the presence of the same Pectobacterium and Dickeya species on asymptomatic vegetables and ornamental plants as those which occurred on plants with disease symptoms in the same countries from which we have analysed samples (Brasil, Egypt, Morocco, UK, USA, Israel, Finland, Norway, the Netherlands and Poland) [3,12,17,18,21,[40][41][42][43][44][45][46][47][48][49][50][51]. In addition, the presence of virulent strains of the same species of Pectobacterium and Dickeya has been described in the rivers of Finland [45] and France [39].
We also observed that different Pectobacterium species can occur on the same asymptomatic plant sample. So far, the co-occurrence of various Pectobacterium species on the same plant samples have been noted on the symptomatic plants only [3]. Based on the obtained results, we can conclude that in the same sample of the plant we did not observe the coexistence of P. brasiliense with P. atrosepticum or P. parmentieri. It can be assumed that the strains belonging to the above-mentioned species may be antagonistic towards each other. It has been experimentally shown that P. brasiliense PBR1692 T produces bacteriocins against P. atrosepticum SCRI1043 and P. carotovorum WPP14 [52]. Indeed, in our observations, we did not observe strains of P. brasilense co-existing on the same plant sample with P. atrosepticum or P. parmentieri strains. Besides bacteriocins, some strains of Pectobacterium, Dickeya and Serratia (all these species were detected by us on asymptomatic plants) produce β-lactam and carbapenem antibiotics that play a role in bacterial competition and might give them better fitness in the ecological niche [53].
Furthermore, some of the tested Pectobacterium strains showed resistance to antibiotics. Under natural conditions, this feature is acquired as a means of protection against bacteriocins secreted by other bacteria with which the genus Pectobacterium competes for ecological niche (e.g., the genus Pseudomonas). Plants also produce bactericidal compounds, e.g., isothiocyanates. Genetic determinants of the enzymes responsible for their degradation have been found in the chromosomes of many Pectobacterium species [20]. Conjugation plasmids are a possible way of acquiring resistance to antibiotics or other bactericidal compounds. In addition, plasmids carry, for example, genes coding heavy metal removal pump systems that allow the survival in the presence of many plant protection compounds. Such plasmids carrying enzymes enabling antibiotic degradation have been described in P. versatile SCC1 and P. zantedeschiae 9M T strains [5,54]. The ability to take in plasmids and acquire new traits provides an advantage for bacteria in adapting to new environmental conditions. We also decided to check if Pectobacterium isolates are susceptible to specific antibiotics. This may pose a serious economic problem in the future. Strains with the Extended Spectrum Betalactamases positive (ESBL+) phenotype are resistant to many β-lactam antibiotics and other chemical compounds, which makes it easier for them to undergo positive selection and persist in flora, and finally spread in the environment. The ESBL coding genes are located on plasmids, which usually also contain genes conditioning resistance to e.g., aminoglycosides, co-trimoxazole, tetracyclines or chloramphenicol [55]. This is currently the most important topic in the field of antimicrobial drug resistance. Build-up of multiresistance carbapenemase-producing microbes provokes questions about the future of treatment of clinical and environmental infections [56,57]. Some of the isolates tested showed resistance or low sensitivity to ampicillin.
In our recent studies, we have shown that bacteria from the genus Pectobacterium produce extracellular membrane vesicles (MVs) harbouring various enzymes, among them β-lactamases. Furthermore, we have shown that MVs produced by ampicillin resistant P. versatile strain DPMP190 enable the growth of sensitive E. coli strain in the presence of 300 µg mL −1 of ampicillin in the medium [58]. The mechanism of secretion of enzymes degrading antibiotics outside the cell via MVs has the key importance of the effective colonisation of an ecological niche and competence with other microorganisms present in it. Thus, the presence on the same plant samples strains of P. versatile that are resistant to antibiotics and produce MVs with active β-lactamases and carbapenemases and might allow for the growth of strains that are susceptible to bacteriocins produced by strains coexisting in the same niche. Furthermore, β-lactamases which can cut the lactam ring are able to degrade the homoserine lactones. Therefore, they can play a significant role in disrupting the cell signalling of other bacteria competing for the same niche, for example, hindering their biofilm formation or reducing virulence. In our observations, P. versatile occurred on the same plant sample together with P. carotovorum, P. brasiliense, P. atrosepticum or with P. parvum. This co-existence could be due to secretion via MVs of enzymes degrading antimicrobial compounds produced by other microorganisms present on the same plant.
Recently, numerous P. versatile strains and limited number of P. brasiliense and P. polaris strains harbouring genetic determinates of beta-lactamases have been described [59]. The large variety of environments from which P. versatile strains carrying β-lactamases have been isolated indicates the extraordinary ability of these bacteria to colonise various environments.
Based on the results of pathogenicity tests, we have demonstrated that strains isolated from asymptomatic plants could macerate plant tissues in laboratory conditions. Furthermore, strains isolated from symptomless plants have a similar ability to macerate plant tissues as strains retrieved from plants with disease symptoms. Except for four strains, all of them were able to macerate plant species other than those from which they have been isolated. Similar observations are widely described in the literature in the case of strains isolated from symptomatic plants or water [3].
However, for the strains from symptomatic plants a slightly smaller rot area was observed than for asymptomatic strains ( Figure 5). The observed difference between mean rotting area of strains isolated from symptomatic and asymptomatic plants is statistically significant; however, the ranges exhibited by both groups of strains are very similar and overlapping. This difference may be due to the fact that the reference strains and Polish strains derived from plants with disease symptoms were isolated much earlier and have been stored in laboratories at least for 25 years; therefore, it cannot be excluded that they have partially lost their virulence. However, to definitively ascertain whether the strains from symptomatic and asymptomatic plants exhibit a statistically significant difference in virulence, additional tests should be carried out on a larger group of strains. Primarily because in the range of each of the tested species, the strains show a very diverse ability to macerate plant tissues.
Undoubtedly, Pectobacterium strains are sensitive to the lack of water in the environment in which they occur. However, they possess the ability to survive in conditions of limited water availability. Some of the strains tested after 48 h of incubation in the condition of limited water availability by adding PEG to the medium were able to resume their growth. This may explain why Pectobacterium is able to survive on the vegetables or ornamental plants in warm climates or during transport and storage. We also checked the survivability of tested bacteria in different pH. The most optimal range of pH for growth of Pectobacterium spp. was between 5 and 6, which is comparable to pH usually occurring in plant tissues [60,61]. They were also able to cope well at pH 7 and 8. After 48 h of incubation, the strains were also able to adjust to pH 10 and 11 and resumed their growth. Most of the strains could not survive in the environment with pH = 4. Bacteria of the genus Pectobacterium grew well in the medium with salinity in the range from 0 to 4%. Above that value, bacteria grew poorly. However, the spot tests indicated that the bacteria remained viable in such extreme conditions. It can be concluded that they were able to arrest their metabolism and survive while awaiting favourable growth conditions. The most resistant to the changes in the pH and salinity conditions was the Moroccan strain P. brasiliense DPMP55 isolated from potato. It is worth underlining that ware potatoes from Mediterranean regions are sold frequently and are readily available in European stores during the winter period. Other stress-resistant strains were two isolates of P. carotovorum DPMP199 and DPMP200 from Egyptian potatoes and two P. versatile isolates from cactus DPMP198 DPMP202. These strains are extremely resistant to the reduced water content in the environment, and thus they have a much greater possibility of surviving long-term transport and storage.
Undoubtedly, the results of adaptation tests and antibiotic resistance indicate that, among the detected Pectobacterium species, strains classified as P. versatile are the most resistant to antibiotics and are characterised by the ability to secrete β-lactamases and carbapenemases. In addition, of all the strains tested, two strains of P. versatile DPMP198 and DPMP202 from the cactus can grow in the most extensive pH range and the lowest water availability, while the P. versatile DPMP402 strain is viable in a medium containing from 0-11% NaCl. Thus, the species P. versatile, characterised by high genetic and phenotypic variability and high adaptation abilities, has the greatest potential to spread and effectively colonise new environments. It is confirmed by the number of strains isolated from various environments, from plants to water, soil, or insects. This taxon is the most numerous among the strains collected in the collections and among species detected on asymptomatic plants, which was shown in this study.

Bacterial Strains and Growth Conditions
Pectobacterium strains used in this study are listed in Table 1 Additionally, 26 strains isolated from symptomatic plants were used as a reference for comparison purposes.
To isolate new bacterial strains, about 15 cm 2 of plant area were suspended in 15 mL of 0.96% NaCl and were homogenised by grinding in a mortar and pestle. After 2 h of preincubation with shaking at 28 • C, the samples were serially diluted. Next, 100 µL aliquots of serial dilutions of homogenate (10 −2 , 10 −3 , 10 −4 and 10 −5 ) were plated on a Crystal Violet Pectate (CVP) medium [62] and incubated for 48 h at 28 • C. Colonies that formed cavities were restreaked on CVP medium and incubated as previously. For long time storage, all isolates were kept as frozen stocks at −80 • C To prepare frozen stocks, single bacterial colonies were transferred to 7 mL of TSB medium and then grown for 48 h at 28 • C with shaking. Next, 500 µL of bacterial culture was mixed with 500 µL sterile 80% glycerol.

Plant Tissue Maceration Assays
The ability to macerate plant tissue was determined for 26 pectinolytic strains newly isolated from asymptomatic plants and 21 strains originating from plants with disease symptoms. For the pathogenicity assays, bacteria of various origins were chosen (Table S1).
Leaves of Irises and Chicory were washed with distilled water. An overnight bacterial culture in the TSB medium was diluted with physiological saline solution to an optical density of 0.5 McF. A one-centimeter cut was made on the surface of the leaf across the conductive bundle, and 25 µL of the bacterial suspension was then dripped into the cut. Inoculated leaves (in three replicates per strain) were placed in plastic sampling bags with sealing strips lined with paper towels soaked in sterile distilled water. Such prepared samples were incubated at 28 • C for 48 h. After 24 and 48 h of inoculation, the average area of rotten tissue was calculated. As a negative control, leaves were inoculated with 25 µL of sterile water.
Potato tubers were thoroughly washed, and their surface was disinfected in a 1% hypochlorite solution bath. The overnight bacterial culture in TSB medium was diluted with Ringer solution to an optical density of 0.5 McF. The tubers were punctured with pipette tips containing 50 µL of the suspension. The tips were left in the tubers. A total of three punctures were made on each tuber. Each strain was tested on 3 tubers in triplicates. Inoculated tubers were placed in plastic boxes. In addition, 500 mL of distilled water was poured into the bottom of the boxes to achieve relative humidity above 90%. After 72 h of incubation at 28 • C, the diameter of the macerated tissue was measured. As a negative control, pipette tips inserted into tubers contained sterile water rather than a bacterial suspension.

Adaptation to Various Environmental Conditions
The ability to grow in various environmental conditions such as different pH, salinity and water availability was determined for 22 pectinolytic strains newly isolated from asymptomatic plants and 13 strains originating from plants with disease symptoms. (Tables 3-5).
The assays were performed in 96-well titration plates. The 200 µL of TSB medium with various pH, salinity and PEG concentration was inoculated with 5 µL of bacterial suspension with an optical density of 0.5 McF. The plates were incubated with shaking at 28 • C. The absorbance readings at 600 nm were made after 0, 6, 24 and 48 h of incubation, using the Infinite M200 Pro (Tecan). The experiments with two replicates were performed twice.
To assess the viability of bacteria under different pH conditions, salinity and water availability after 48 h of incubation, spot tests were performed on TSA medium. Furthermore, 5 µL of bacterial culture was withdrawn from each well and dropped onto a plate, which was then incubated for 48 h at 28 • C. Additionally, after performing the spot test, 20 µL of 0.02% resazurin solution was added to each well, and the plate was incubated for 24 h at 28 • C. The result was read colorimetrically. During bacterial metabolism, purple resazurin is transformed to pink resorufin (pH = 6.5). Under acidic conditions, resorufin takes on a yellow colour (pH = 3.8). This allows for a colorimetric reading of the presence of acid metabolism products in the medium.

Antibiotic Susceptibility Assay
The antibiotic susceptibility of 34 selected Pectobacterium strains (31 isolated from asymptomatic plants and 3 from symptomatic) was tested by a standard disc diffusion method. In addition, 100 µL of a bacterial suspension with an optical density of 0.5 McF was spread on a Mueller-Hinton (MH) medium. Antibiotic discs containing ampicillin 10 µg, erythromycin 15 µg, gentamicin 250 µg, kanamycin 30 µg, streptomycin 10 µg, tetracycline 15 µg were then applied (Emapol). The plates were incubated for 24 h at 28 • C. Next, the zone of bacterial growth inhibition around the antibiotic disc was assessed.
Moreover, for strains that showed resistance to at least one antibiotic in the disc diffusion test, the production of beta-lactamases and carbapenemases was determined with application of two chromogenic media, ESBL (extended-spectrum beta-lactamases) chromagar and KPC (Klebsiella pneumoniae carbapenemase) plates (GRASSO). The control Escherichia coli NCTC 13351 and Klebsiella pneumoniae BAA-1705, which grow on the abovementioned media and have ESBL and KPC resistance mechanisms, have been used.

Statistical Analysis
The analysis of differences between the groups was performed using the ANOVA with Welch corrections for nonhomogeneous variances criterion followed by a post-hoc Games-Howell analysis in R [63].

DNA Isolation
For DNA isolation, bacterial strains were grown overnight in 7 mL of Tryptic Soy Broth (TSB) at 28 • C with shaking. Cells were harvested by centrifugation and resuspended in 500 µL TE buffer (50 mM Tris/HCl, 40 mM EDTA, pH 8.0). Afterwards, the cell lysis and nucleic acids extraction were carried out according to the protocol proposed by a Joint Genome Institute for bacterial DNA isolation using CTAB [64] followed by the RNA digestion using Turbo RNase (Ambion). DNA quantity and quality were assessed first using a NanoDrop Spectrophotometer and later with agarose gel electrophoresis.

PCR Amplification Sequencing and Phylogenetic Analysis
DNA amplification was performed in 25 µL reaction volumes using PCR Master Mix (Thermo Scientific; K0171) according to the manufacturer's instruction. Amplification was performed using a T100 Bio-Rad thermocycler. The amplified products were separated in 1.5% (w/v) agarose gel at 100 V for 40 min in 0.5xTAE buffer and visualised with UV light after staining in ethidium bromide (0.5 g mL −1 ). Species specific PCR reactions were performed according to the previously described protocols [29][30][31][32]35]. The genetic diversity of Pectobacterium and Dickeya strains was analysed with a fingerprinting method, Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR), according to the procedure described by Versalovic et al. [34]. For amplification and sequencing of the recA gene fragment, primers previously described were used [38]. Sequencing was carried out using an ABI PRISM DNA Sequencer (PerkinElmer) according to the manufacturer's manual. Both strands were sequenced using the forward and reverse PCR primers.
Furthermore, the obtained sequences were subjected to BLAST sequence similarity search analysis to identify the nearest taxa. The obtained sequences of the recA gene and sequences of the closest representative taxa that belong to Pectobacterium and Dickeya were aligned using an MAFFT algorithm in Geneious v9.1.8. [65]. The phylogenetic analysis was performed with the MEGA v. X software, (www.megasoftware.net, accessed on 20 May 2022), and trees were constructed using the Maximum Likelihood algorithm and Hasegawa-Kishino-Yano models selected on the model test module implemented in MEGA. Bootstrap analysis with 1000 replications was performed to assess the robustness of the clusters.

Conclusions
Our research proved that bacteria of the genus Pectobacterium isolated from asymptomatic ware vegetables and ornamental plants can cause maceration of plant tissues of more than one plant species. These strains were also able to adapt to extreme environmental conditions, such as low water accessibility, acidic and alkaline pH, and high salinity. The antibiograms' results showed that some of these bacteria had resistance to betalactams, macrolides, aminoglicosides and tetracyclines; in addition, they might possess beta-lactamases of extended spectrum or carbapenemases.
Undoubtedly, we have demonstrated that Pectobacterium strains isolated from symptomless vegetables and ornamental plants traded internationally show a high potential for adaptation to adverse environmental conditions and ability to change the host plant. As a result, they may contribute to the success of the genus Pectobacterium and accelerate its spreading between different climatic zones and facilitate the colonisation of different ecological niches.
The most important claim we want to make, which has not been previously described, is that internationally traded ware vegetables, ornamental plants and herbs which do not undergo strict phytosanitary control serve as a significant transmission medium responsible for global distribution of widely observed species such as P. versatile, which remains the most frequently isolated pathogenic taxon from Pectobacterium genus. Thus, we would like to bring the attention of the scientific community to this underexplored area, and look beyond pathogen presence in seed material, towards mass market wares and organic waste which become more and more important in the age of sustainable agriculture and a zero waste lifestyle.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pathogens11070728/s1, Table S1: Results of pathogenicity tests. Table S2: Calculations of statistical significance; Figure S1 Comparison of the potato tissue maceration ability; Figure S2: Comparison of the chicory tissue maceration ability.