Ralstonia solanacearum (phylotype II) isolated from Rosa spp. in the Netherlands is closely related to phylotype II isolates from other sources in the Netherlands and is virulent on potato

Ralstonia solanacearum (phylotype II) isolated from Rosa spp. in the Netherlands is closely related to phylotype II isolates from other sources in the Netherlands and is virulent on potato | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ralstonia solanacearum (phylotype II) isolated from Rosa spp. in the Netherlands is closely related to phylotype II isolates from other sources in the Netherlands and is virulent on potato Nathalie Blom, Peggy Gorkink-Smits, Marco Landman, Jeroen van de Bilt, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4396851/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Oct, 2024 Read the published version in European Journal of Plant Pathology → Version 1 posted 6 You are reading this latest preprint version Abstract In 2018, during an annual survey in the Netherlands, Ralstonia solanacearum phylotype II (phy II) was found in asymptomatic greenhouse rose plants at three geographic locations. These findings were remarkable, since previous findings of Ralstonia sp. in rose always concerned Ralstonia pseudosolanacearum phylotype I (phy I). Therefore, no information was available on the virulence of R . solanacearum phy II on rose. In this study, R . solanacearum phy II isolates PD 7421 and PD 7394, isolated in 2018 from asymptomatic ornamental rose ( Rosa spp.), were assessed for their virulence in two rose cultivars (“Armando” and “Red Naomi”) at two temperatures. No typical symptoms were observed for PD 7421 and PD 7394 on the two rose cultivars, irrespective of the temperature. However, latent infections upon inoculation of these isolates on rose did occur. R . solanacearum phy II is known as a major potato pathogen, where it causes brown rot. Whole genome multilocus sequence typing analysis demonstrated that the R . solanacearum phy II isolates from rose were closely related to R . solanacearum phy II isolates previously found in seed potato and surface water in the Netherlands. Because of this close genetic relatedness, the virulence of PD 7421 and PD 7394 was also assessed in potato plants, where both isolates caused severe disease symptoms on the shoots as well as the daughter tubers. This implies that rose can act as a reservoir for R . solanacearum phy II and, in this way, can potentially be involved in spreading this bacterium. Ralstonia solanacearum phylotype II Rosa spp. Solanum tuberosum virulence phylogenetic analysis Figures Figure 1 Figure 2 Figure 3 Introduction Ralstonia solanacearum is considered to be one of the most important plant pathogenic bacteria, due to its broad host range and the economic losses it causes, especially in potato (Mansfield et al., 2012 ). In the past R. solanacearum was considered a species complex (RSSC), composed of a heterogenous group of strains differing in host range and geographic origin (Mansfield et al., 2012 ), and therefore, its members have been classified in several different ways. Examples are division into races (Buddenhagen & Kelman, 1964 ), into biotypes (Hayward, 1964 ) and more recently into four phylotypes, which are monophyletic clusters of strains that roughly correlate with the geographic origin of the isolates within these clusters (Prior & Fegan, 2005 ). In 2014 the RSSC was split into three separate bacterial species based on gene sequences and DNA-DNA hybridization data. These species are R. solanacearum (phy II), Ralstonia pseudosolanacearum (phy I and III) and Ralstonia syzygii (phy IV), of which the latter has been divided into three subspecies (Safni et al., 2014 ). All species and subspecies of the former species complex have a quarantine status within the European Union, except for R . syzygii subsp. syzygii (EU, 2019). Bacteria within the RSSC are soil-borne vascular pathogens. They colonize the xylem vessels of the plant, after which they spread systemically through their host. Accumulation of cells in the vascular tissue blocks the vessels and thereby impedes the water flow, leading to wilting symptoms (Lowe-Power et al., 2018 ). Members of the RSSC are highly infectious and are able to survive in water (Alvarez et al., 2008 ; Stevens et al., 2018 ; Van Elsas et al., 2001 ; Wenneker et al., 1998 ), soil (Van Elsas et al., 2000 ), and even on surfaces, including wood, rubber and metal (Wenneker et al., 1998 ). It is known that the weed bittersweet ( Solanum dulcamara ) can get infected when it is in contact with contaminated surface water (Wenneker et al. 1999; Vogelaar et al., 2022). After infection, the bacteria can survive in these plants for years and can leak back into the water, allowing for recontamination of surface water after cold winters (Elphinstone et al. 1998). In addition to their broad host range, high infectiousness and ability to survive in different environments, another difficulty in preventing members of the RSSC to spread, is their frequent occurrence as latent infections. For example, R . solanacearum can be latently present on the surface, in the lenticels and in the vascular tissue of potato tubers (Sunaina et al., 1989 ), which poses a risk of spreading the disease without being noticed. An extensive outbreak of R. solanacearum (phy II, race 3, biovar 2) in potato occurred in the Netherlands in 1995. In that year, 94 farms were found to be infected with the bacterium (Schans & Steeghs, 1998 ). Most of these farms were either directly infected by using contaminated surface water for irrigation, or the infected lots were clonally propagated from seed potatoes of the cultivar ‘Bildtstar’ that had been irrigated with infected surface water in previous years (Janse, 2012 ; Schans & Steeghs, 1998 ). The control strategy focused on elimination of R . solanacearum from the entire potato production chain and prevention of new horizontal introductions into it (Schans & Steeghs, 1998 ). This approach has proven to be effective, in the fifteen years after the outbreak, the number of infected potato samples decreased substantially (Janse, 2012 ). The current status of R . solanacearum in the potato production chain in the Netherlands is that it is transient, actionable with incidental findings (EPPO Reporting Service no. 01–2017). In 2015, there has been a major outbreak of R. pseudosolanacearum in Rosa spp. in the Netherlands. Greenhouse roses showed wilting symptoms, leaf chlorosis, early leaf drop, necrosis of pruned branches and in some cases bacterial slime protruding from the stem. Until then, rose was not known to be a host of any of the members of the RSSC (Tjou-Tam-Sin et al., 2017b ). The causal agent was identified as R . pseudosolanacearum phy I. Phylogenetic assignment based on amplified fragment length polymorphism data (AFLP) and multi locus phylogeny (MLSA) led to the conclusion that the R. pseudosolanacearum phy I isolates from rose clustered together in one monophyletic group that contained, besides the isolates from rose, only one other isolate which originated from eggplant in India (Bergsma-Vlami et al., 2018 ; Tjou-Tam-Sin et al., 2017b ). This suggested a single and recent introduction of R . pseudosolanacearum phy I into the rose industry in the Netherlands. In the years 2016–2017 R. pseudosolanacearum (phy I) was also found in greenhouse rose cultivation in other European countries (EPPO reporting service, articles 2016/039, 2017/018, 2017/085, and 2017/172). In 2017 similar symptoms as the ones in greenhouse roses in the Netherlands, were observed in cultivated roses in Korea (Kim et al., 2019 ). Kim et al. ( 2019 ) were able to isolate R. pseudosolanacearum (phy I) from rose plants from three different regions in Korea by plating and confirming the identity of suspect colonies with a phylotype-specific PCR. For a better understanding of the behavior of R. pseudosolanacearum (phy I) on this new host plant, Tjou-Tam-Sin et al. ( 2017a ) studied the effect of different factors on the virulence of these isolates on rose. They showed that rose cultivars differed in their susceptibility to R . pseudosolanacearum (phy I) and that a higher temperature resulted in a higher disease incidence and disease severity (Tjou-Tam-Sin et al., 2017a ). In 2018, during an annual survey in the Netherlands, Ralstonia sp. was found in rose plants again. This time Ralstonia sp. was isolated from asymptomatic plants. Immunofluorescence, real-time PCR, fatty acid analysis, sequence analysis ( egl and mut S), MALDI-TOF MS and a pathogenicity test were performed to identify the isolates. Remarkably, analysis of the egl and mut S loci and MALDI-TOF MS revealed that five of these isolates, originating from three different geographic locations, did not belong to phylotype I, which had been consistently found in rose since 2015, but to phylotype II. This phylotype II has never been found in rose before and therefore there is currently no information available on the virulence of R. solanacearum (phy II) on rose. The aim of this study was to determine the virulence of R. solanacearum phy II, isolated from asymptomatic Rosa spp., on rose plants, under various conditions. Virulence was assessed using two different rose cultivars at two different temperatures. A whole genome multilocus sequence typing (wgMLST) analysis was additionally performed to determine the genetic relatedness of R. solanacearum phy II isolated from symptomless Rosa spp. with R. solanacearum (phy II) from potato, surface water, soil and other host plants previously found in the Netherlands. Furthermore, the virulence of the R. solanacearum phy II strains isolated from asymptomatic Rosa spp., was also assessed in potato plants, given the previous findings of R. solanacearum phy II in seed potato and surface water in the Netherlands. Materials & Methods Bacterial isolates Two R. solanacearum phy II isolates, PD 7421 and PD 7394, that have been isolated from asymptomatic rose plants during an annual survey in 2018, were selected for inoculation of rose and potato plants (Table 1 ). In addition to these two isolates, an R. solanacearum phy II isolate from potato was included in the inoculation experiments, as well as an R . pseudosolanacearum phy I isolate from rose, which functioned as a positive control (PC; Table 1 ). Lyophilized bacterial cultures were transferred to nutrient agar (NA) broth and were placed in a shaker to incubate at room temperature for 0.5-1 hour. After incubation, the liquid medium was plated onto the general medium yeast peptone glucose agar (YPG). After two days, the bacterium was transferred to NA from a single colony. Two-day-old pure cultures were used to prepare the bacterial suspensions used in the inoculation experiments. Table 1 Ralstonia isolates used in the stem inoculation experiments on rose and potato plants. Isolate External collection number(s) Ralstonia species Phylotype Host of origin Geographic origin Year of isolation PD 2762 N/A R. solanacearum II Solanum tuberosum cv. “Bildtstar” The Netherlands 1995 PD 7394 N/A R. solanacearum II Rosa sp. cv. “GL” The Netherlands 2018 PD 7421 N/A R. solanacearum II Rosa sp. cv. “Lucky Red” The Netherlands 2018 PD 7123 CFBP 8587 R. pseudosolanacearum I Rosa sp. cv. “Red Naomi” The Netherlands 2015 Stem inoculation experiment in Rosa sp. Rose plants of cv. “Armando” and cv. “Red Naomi” were inoculated with a bacterial suspension (± 10^8 cfu/mL) or with sterile 0.01 M phosphate buffer (PB) as a negative control (NC). Inoculations were performed as described by Tjou-Tam-Sin et al. 2017a . Plants of cv. “Armando” were inoculated 3 weeks after pruning. New shoots were 0,5 − 1 cm thick at the moment of inoculation, and per plant 3 shoots were inoculated. Plants of cv. “Red Naomi” consisted of only one shoot, that was inoculated one week after pruning the flower. The experiment was conducted at a temperature of 20°C and 26°C, a relative humidity of 85% and a 14h/10h day/night cycle. For every combination of temperature and inoculum, 4 plants of each rose cultivar were inoculated. Disease severity was scored on a scale from 0–3 until 106 dpi, where 0 = healthy plants, 1 = mild symptoms (leaflets of compound leaves are hanging down and/or leaf veins start to yellow on one or a number of leaves, mainly at the bottom of the plant) 2 = severe symptoms (expansion of yellowing leaf veins, chlorotic and necrotic leaves, early leaf drop) and 3 = plant death, based on the scale described by Tjou-Tam-Sin et al. ( 2017a ). Re-isolations were performed on all inoculated rose plants, either during the experiment or at the end of the experiment (at 106 dpi), in case of no symptom development. For re-isolations, plant extracts were made from pieces of petiole from the upper part of the plant (in case of symptomatic plants) or stem pieces from above the inoculation points of an inoculated stem (in case of asymptomatic plants). Symptomatic material was placed in 5 mL phosphate buffer saline (PBS) 0.01 M and incubated at room temperature for 30 minutes. After incubation, 50 µl extract was plated onto 6 SMSA plates by means of dilution plating. For asymptomatic plants, 15 stem pieces of ± 1.5 cm of an inoculated branch were externally disinfected, crushed, and, after adding 7–10 mL PB 0.05M, placed in a shaker for 30 minutes at 100 rpm at RT. Extract was centrifuged for 15 minutes at 7000 g. Supernatant was discarded and 1,5 mL PB 0.01M was added. Then, 50 µL was plated onto 6 SMSA plates by means of dilution plating. After 6 days of incubation at 28 o C, plates were checked for typical colonies. Identity of these colonies was confirmed by MALDI-TOF MS (Van de Bilt et al., 2018 ). Phylogenetic analysis Phylogenetic analysis was performed with the 23 isolates shown in Supplemental Table 1. Purified cultures were used to isolate DNA using the High Pure PCR Template Preparation Kit (Roche, according to the manufacturer’s instructions, except for the centrifugation step, which was performed at 6000 g instead of 8000 g. DNA sequencing was performed at GenomeScan (Leiden, the Netherlands) for Illumina 150PE (paired-end) sequencing using the NovaSeq 6000 with at least 2Gb output per sample. Samples were processed using the NEBNext® Ultra II FS DNA module and the NEBNext® Ultra II Ligation module. Fragmentation, A-tailing and ligation of sequencing adapters and PCR using NEBNext® Ultra II Q5 master mix of the resulting product was performed according to the procedure described in the NEBNext Ultra II FS DNA module and NEBNext Ultra II Ligation module instruction manual. After preparation, the quality and yield for all samples was measured with the Fragment Analyzer (Agilent Technologies, USA). The obtained read files, were deposited under BioProject accession PRJNA1089092. Illumina paired-end read quality was checked using fastqc ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). Subsequently, quality and adaptor trimming was performed using Trimmomatic v 0.39, with a sliding window of 5:30 and a minimum length at 50 nucleotides (Bolger et al., 2014 ). Trimmed reads were input for a de novo assembly using Spades v3.13.0 (Bankevich et al., 2012 ; Prjibelski et al., 2020 ). Assemblies were annotated using the NCBI prokaryotic genome annotation pipeline (PGAP version 6.5) (Tatusova et al., 2016 ) and have been deposited at DDBJ/ENA/GenBank under the accessions indicated in Supplemental Table 1.Whole genome alignments were made with the Whole Genome Alignment plugin in CLC Genomics Workbench 21.0.5 (Qiagen, Aarhus, Denmark). Next, ANI comparisons were generated using the same plugin. From these ANI scores unweighted pair group method with arithmetic mean (UPGMA) trees were generated. Contig assemblies were utilized for a whole genome Multi Locus Sequence Typing (wgMLST) analysis using ChewBBACA v2.8.4 (Silva et al., 2018 ). To perform a wgMLST on the selected Ralstonia strains, first a training file was created for strain PD7642 using prodigal (Hyatt et al., 2010 ). Subsequently, the training file and 106 R. solanacearum genomes (Supplemental Table 2) were used to create a wgMLST scheme facilitated by the CreateSchema command in ChewBBACA. After creating the scheme, the wgMLST allele calling was performed for the 12 closely related isolates (Silva et al., 2018 ). Visualization of the wgMLST results was performed by Phyloviz v2.0 (Nascimento et al., 2017 ). In total 4343 loci were included in the wgMLST analysis. Each isolate was compared with all other isolates included in the analysis. For each combination of two isolates, the number of loci with > 1 SNP relative to each other was calculated and these numbers were used to create a minimum spanning tree. In the minimum spanning tree, the nodes (isolates) are connected by edges associated with numbers representing the number of loci with SNPs, in a way that every node is included and that the sum of the edges is as low as possible. Stem inoculation experiment in potato Potato plants of cv. “Saphir” were inoculated with a bacterial suspension (± 10^8 cfu/mL) or with 0.01 M PB as an NC. Plants were inoculated 18 days after planting the tubers, when plants were ± 30 cm high. Per plant, 3 stems were inoculated at two different heights, just above and 10 cm above the soil level, using a syringe with a 0.5 x 16 mm needle. Per isolate 10 plants were inoculated. Plants were kept at 20°C (day and night) and a relative humidity of 60%. Disease severity was scored on a scale from 0–3, for a period of 6 weeks. Plants were scored as 0 when there were no symptoms, as 1 when symptoms were only present on inoculated stems, as 2 when symptoms were also present on non-inoculated stems and as 3 when plants were dead. Furthermore, the total number of daughter tubers (> 1 cm) and the number of symptomatic daughter tubers per plant were counted. Re-isolations were performed on the above ground tissue of all plants at the end of the experiment, or during the experiment in case of heavily infected plants. Re-isolations were also performed on the newly formed tubers, as previously described (Overeem et al., 2023 ). For this, a combined sample was taken per plant, containing pieces of the vascular bundle of each daughter tuber, regardless of symptom development. The experiment was repeated using the potato cultivar “Talent”. Identification of re-isolates with MALDI-TOF MS Colonies isolated from the inoculated rose and potato plants with a typical R . ( pseudo ) solanacearum morphology were analyzed on a Bruker MALDI Biotyper using the direct transfer method as described previously (Van de Bilt et al., 2018 ). The obtained spectra were matched against the Bruker MSP library and against an in-house created MSP library (Van de Bilt et al., 2018 ) that includes MSPs of all four RSSC phylotypes. Scores > 2.00 are considered as a positive identification. Statistics Statistical analyses were performed using R (version 4.1.2) and the dunn. test package (version 1.3.5). The area under the disease progress curve (AUDPC) was calculated as described by Madden et al. ( 2007 ). Averages of the AUDPC scores and percentage of daughter tubers with symptoms were compared using a Kruskal-Wallis rank sum test. Pairwise comparisons were performed using the Dunn’s test with a Hochberg correction of the p-value. For the number of daughter tubers an ANOVA followed by a Tukey multiple comparison of means test was performed. Results Ralstonia solanacearum phy II from rose causes latent infections in rose To investigate the virulence of R. solanacearum phy II isolated from asymptomatic rose plants during the annual survey in 2018, isolates PD 7421 and PD 7394 were inoculated in rose plants of cultivars “Armando” and “Red Naomi” at two different temperatures. Both isolates did not cause any typical symptoms on rose regardless of the cultivar or temperature (Supplemental Fig. 1), resulting in an AUDPC of 0 after 106 days (Table 2 ). Also the potato R. solanacearum (phy II) isolate PD 2762, did not cause any typical symptoms on rose in any of the treatments (Supplemental Fig. 1). In contrast, the R. pseudosolanacearum phy I isolate PD7123, that is known to be highly virulent on rose, did cause symptoms (Supplemental Fig. 1) and resulted in an AUDPC ranging between 32.6–91.9 depending on the rose cultivar and temperature (Table 2 ). Symptom development upon inoculation with PD7123 first started with leaflets of the compound leaves hanging down and/or yellowing of the veins in the leaves. After this, chlorosis expanded from the veins of the leaves to the whole leaf surface, and chlorotic leaves eventually turned necrotic. Sometimes, but not always, affected leaves fell off. Table 2 Disease severity, expressed as area under the disease progress curve (AUDPC), and proportion of highly infected plants of rose cultivars “Armando” and “Red Naomi” after stem inoculation with different Ralstonia isolates at 20°C and 26°C at 106 dpi. Isolate Rose cultivar AUDPC 20°C AUDPC 26°C Proportion highly infected plants a 20°C Proportion highly infected plants a 26°C PD 7123 Armando 59 73.8 4/4 4/4 Red Naomi 32.6 91.9 4/4 4/4 PD 2762 Armando 0 0 3/4 b 3/4 b Red Naomi 0 0 2/4 b 3/4 b PD 7421 Armando 0 0 3/4 b 4/4 b Red Naomi 0 0 0/4 b 2/4 b PD 7394 Armando 0 0 3/4 b 2/4 b Red Naomi 0 0 2/4 b 4/4 b NC Armando 0 0 0/4 b 0/4 b Red Naomi 0 0 0/4 b 0/4 b a Plants from which > 200 typical Ralstonia colonies were re-isolated. b Latent infection Since R. solanacearum phy II isolates PD 7421 and PD 7394 were isolated from asymptomatic plants, all inoculated rose plants were checked for the presence of these bacteria as latent infection, at the end of the experiment (106 dpi). In almost every treatment at least two of the four inoculated plants were found to be latently infected when re-isolations were performed, the only exception was “Red Naomi” inoculated with PD 7421 at 20°C (Table 2 ). So, despite the lack of symptoms the pathogen is able to multiply inside the rose plants. Ralstonia solanacearum phy II isolates from rose and other sources in the Netherlands show a close genetic relationship The genetic relatedness between the R. solanacearum phy II isolates found in rose in 2018, PD 7421 and PD 7394, and 12 other R. solanacearum phy II isolates, originating from surface water, soil and different host plants, including potato, was investigated. For this, a whole genome alignment using 23 Ralstonia isolates (Supplemental Table 1) and pair-wise comparisons based on average nucleotide identity (ANI) scores were performed (Fig. 1 A). The majority of the R. solanacearum phy II isolates included in the analysis clustered together in one monophyletic group with hardly any variation, this cluster contained only isolates originating from the Netherlands. Only two R. solanacearum phy II isolates, one from Ecuador (PD 3222- Anthurium andreanum ) and one from Brazil (PD 1414- Solanum tuberosum ), were placed outside this cluster and, with ANI scores of 97.6 and 96.4, respectively, were more distantly related to the 12 Dutch R. solanacearum phy II isolates. The dendrogram based on ANI values is not discriminative enough to show small genetic differences between the 12 R. solanacearum phy II isolates found in the Netherlands. Therefore, a whole genome multilocus sequence typing (wgMLST) analysis was performed and a minimum spanning tree was created (Fig. 1 B). The four R. solanacearum phy II isolates from rose (numbers 1–4) are very closely related to each other, and with a maximum number of genes with at least one SNP of 18 between number 2 (PD 7397) and number 3 (PD 7414, Supplemental Table 3), they might be considered clonally related. The isolates from rose are also very closely related to the three R. solanacearum phy II isolates from surface water in the Netherlands (numbers 5–7) and to R. solanacearum phy II isolate PD 2762 (number 13), which was isolated from potato in 1995. The number of genes with at least one SNP between a R. solanacearum phy II isolate from rose and an R. solanacearum phy II isolate from surface water ranged between 41 and 60 (Supplemental Table 3). Within the R. solanacearum phy II surface water isolates group, this number ranged from 22 to 34. Between the R. solanacearum phy II rose isolates and the R. solanacearum phy II potato isolate, this number ranged from 86 to 91. With a number of genes with at least one SNP ranging from 250 to 501, the group of R. solanacearum phy II isolates on the top left (numbers 8 and 10–12), isolated from different crops and soil, are more distantly related to the four R. solanacearum phy II isolates from rose (Supplemental Table 3), although they can still be seen as closely related isolates. Together these results show that the R. solanacearum phy II isolates from rose are very closely related to the R. solanacearum phy II isolates that have been present in the Netherlands for years, if not decades. Ralstonia solanacearum phy II isolates from rose are virulent on potato To test the virulence of the R. solanacearum phy II isolates from rose on potato, two potato cultivars, “Saphir” and “Talent”, were inoculated with isolates PD 7394 and PD 7421 at a temperature resembling the temperate climate (20°C). Additionally, plants were inoculated with R. solanacearum phy II isolate PD 2762 which is known to be highly virulent on potato and the R . pseudosolanacearum phy I isolate PD 7123 from rose (Table 1 ) which has been shown to be less virulent on potato at lower temperatures. Figure 2 a shows symptom development in the above ground potato tissue of cv. “Saphir” over a period of 41 days. Cv. “Saphir” plants inoculated with R. solanacearum phy II, showed the first symptoms at 8 dpi. In plants inoculated with R . pseudosolanacearum phy I, the first symptoms appeared several days later, at 11 dpi. From 11 dpi onwards, plants inoculated with R. solanacearum phy II started to show severe disease symptoms, as wilting was visible in the non-inoculated stems. At the end of the experiment, at 41 dpi, all cv. “Saphir” potato plants inoculated with either R. solanacearum phy II from potato, or R. solanacearum phy II from rose, showed severe disease symptoms or had died. In contrast, cv. “Saphir” plants inoculated with R . pseudosolanacearum phy I had only developed mild symptoms or no symptoms at all at 20°C at 41 dpi. The AUDPC for cv. “Saphir” plants inoculated with any of the R. solanacearum phy II isolates was significantly higher than for plants inoculated with R . pseudosolanacearum phy I or the NC (Fig. 2 B). However, no significant differences were found in the AUDPC between cv. “Saphir” plants inoculated with R. solanacearum phy II from rose and plants inoculated with R. solanacearum phy II from potato (Fig. 2 b). When the experiment was repeated with cv. “Talent”, in all treatments, including R . pseudosolanacearum phy I, the first mild symptoms appeared at 8 dpi (Fig. 2 c). After 10 days, also the non-inoculated stems started to wilt in cv. “Talent” plants inoculated with any of the R. solanacearum phylotype II isolates. For R . pseudosolanacearum phy I, severe symptoms did not appear before day 15. Similarly to cv. “Saphir”, all cv. “Talent” plants inoculated with either R. solanacearum phy II from potato, or R. solanacearum phy II from rose showed severe symptoms or had died at 42 dpi. Plants of cv. “Talent” inoculated with R . pseudosolanacearum phy I did not reach the stage of plant death, however all plants did develop some symptoms (Fig. 2 c). Similar to cv. “Saphir” also for cv. “Talent” the AUDPC was higher for the three R. solanacearum phy II isolates than for the R . pseudosolanacearum phy I isolate or the NC (Fig. 2 d). However, while the AUDPC for the R. solanacearum phy II isolates PD 2762 (potato) and PD 7394 (rose) were significantly higher than the AUDPC for the R . pseudosolanacearum phy I PD 7123 (rose) and the NC, due to higher variability the difference between the AUDPC for the rose isolates PD 7421 ( R. solanacearum phy II) and PD 7123 ( R . pseudosolanacearum phy I) was not significant. Again, no significant difference was found in the AUDPC between the R. solanacearum phy II isolates from rose and the R. solanacearum phy II isolate from potato (Fig. 2 d). Inoculation with Ralstonia solanacearum phy II isolates from rose leads to decreased daughter tuber formation To test the effect of inoculation with R. solanacearum phy II isolates from rose on tuber formation in potato, the number of daughter tubers formed were counted per potato plant. Figures 3 a and 3 b show the number of daughter tubers formed at 20°C for plants of cv. “Saphir” and cv. “Talent” inoculated with the different bacterial isolates at 41 and 42 dpi, respectively. All R. solanacearum phy II isolates, from both rose and potato, greatly reduced the number of daughter tubers formed compared to the NC. In contrast, this reduction in daughter tuber formation was not seen for R. pseudosolanacearum phy I. This result was valid for both cv. “Saphir” and cv. “Talent”. Inoculation of potato plants with Ralstonia solanacearum phy II isolates from rose leads to symptom development in daughter tubers Not only the number of daughter tubers formed, but also the number of symptomatic daughter tubers present in potato plants inoculated with R. solanacearum phy II isolates, from both rose and potato, or with R. pseudosolanacearum phy I from rose was counted per individual potato plant. Plants of cv. “Saphir” inoculated with R. solanacearum phy II from rose either formed no daughter tubers at all, or formed tubers of which at least one daughter tuber showed disease symptoms, irrespective of the rose R. solanacearum phy II isolate used. The same was valid for cv. “Saphir” plants inoculated with the R. solanacearum phy II isolate from potato (PD 2762). In contrast, only one plant of the cv. “Saphir” inoculated with R. pseudosolanacearum phy I from rose formed at least one daughter tuber with symptoms, while all other cv. “Saphir” plants only formed asymptomatic daughter tubers (Fig. 3 c). Also the percentage of symptomatic daughter tubers was higher for cv. “Saphir” plants inoculated with R. solanacearum phy II, from either rose or potato, than for cv. “Saphir” plants inoculated with R. pseudosolanacearum phy I (Fig. 3 e). All plants of cv. “Talent” inoculated with R. solanacearum phy II from potato formed at least one symptomatic daughter tuber, in case daughter tubers were formed at all. Also plants of cv. “Talent” that were inoculated with one of the R. solanacearum phy II isolates from rose and formed daughter tubers, formed at least one symptomatic daughter tuber, with the exception of one plant inoculated with PD 7421, which only formed asymptomatic daughter tubers. In contrast with cv. “Saphir”, all cv. “Talent” plants inoculated with R. pseudosolanacearum phy I formed at least 1 symptomatic daughter tuber (Fig. 3 d). The mean percentage of symptomatic daughter tubers, however, appears lower for plants inoculated with R. pseudosolanacearum phy I than for plants inoculated with any of the R. solanacearum phy II isolates, although this trend was found not to represent a significant difference (Fig. 3 f). Discussion Since the outbreak of R . pseudosolanacearum phy I in rose in 2015, annual surveys have been conducted in the greenhouse cultivation of rose in the Netherlands. The finding of the R. solanacearum phy II isolates in asymptomatic rose plants in 2018, however, was unexpected, because until then the Ralstonia isolates found in rose always belonged to R. pseudosolanacearum phy I (Bergsma-Vlami et al., 2018 ; Tjou-Tam-Sin et al., 2017b ). Although R. solanacearum phy II was known to cause disease in other crops, for example brown rot in potato, rose was not known to be a natural host of R. solanacearum phy II at that time. Stem inoculation of the rose cultivars “Armando” and “Red Naomi” with two of the R. solanacearum phy II isolates from rose, PD 7394 and PD 7421, did not lead to symptom development on these rose plants at 20°C, nor at 26°C (Table 2 ). This lack of symptom development is in line with the origin of the isolates, i.e. asymptomatic rose plants. Only two rose R. solanacearum phy II isolates were inoculated in the rose plants, but similar results are expected for the other R. solanacearum phy II isolates from the findings in rose in 2018 (Supplemental Table 1), considering their great genetic similarity, as confirmed by the phylogenetic analysis (Fig. 1 a and 1 b). How rose plants respond to inoculation with more distantly related R. solanacearum phy II isolates, for instance the isolates originating from Ecuador and Brazil (Fig. 1 a), remains unknown. Although R. solanacearum phy II PD 7394 and PD 7421 were not able to cause disease symptoms on the rose plants, re-isolations from inoculated rose plants resulted in high numbers of typical Ralstonia colonies in almost every treatment (Table 2 ). This indicates that these R. solanacearum phy II isolates are able to survive and multiply inside the rose plants. Latent infections of R. solanacearum phy II do occur in other plant species as well, including potato (Priou et al., 2001 ; Silveira et al., 2007 ; Sunaina et al., 1989 ), tomato (Grimault & Prior, 1993 , 1994 ; Gutarra et al., 2017 ), pepper (Grimault & Prior, 1994 ), eggplant (Grimault & Prior, 1994 ; Gutarra et al., 2017 ) and geranium (Swanson et al., 2007 ; Swanson et al., 2005 ). Upon inoculation in rose, latent infections with R . solanacearum phy II originating from plant species other than rose have been reported before by Tjou-Tam-Sin et al. ( 2017a ). Similar to our results, inoculation with all R. solanacearum phy II isolates led to a disease incidence of 0%, but in all cases latent infections were confirmed (Tjou-Tam-Sin et al., 2017a ). Latent infections in rose might not lead to visible damage to the rose plants themselves, and thus do not cause any obvious economic damage. However, these infections do form a risk for other crops, as unseen spread of the bacteria to other areas and crops can easily occur. In the past, spread of R . solanacearum phy II through latently infected plant material occurred, for instance, when geranium cuttings were imported from Kenya, Guatemala and Costa Rica into the US (Swanson et al., 2005 ). A similar event occurred in Europe when Pelargonium cuttings from Kenya, that were infected with R . solanacearum phy II, were imported into European countries (Janse et al., 2004 ). Since the Netherlands is the biggest producer of certified seed potato in Northwestern Europe (Goffart et al., 2022 ) and there has been an extensive outbreak of R . solanacearum phy II in potato in the past (Schans & Steeghs, 1998 ), it was highly relevant to test how potato plants respond to inoculation with the R. solanacearum phy II isolates from rose. In contrast to what was seen in the inoculation experiment on rose, the R. solanacearum phy II isolates from rose were highly virulent on potato plants at 20°C (Fig. 2 ). Actually, the R. solanacearum phy II isolates from rose were just as harmful on potato as the R. solanacearum phy II isolate, PD 2762, isolated from potato in the Netherlands in 1995, that was included as a positive control (PC) for the inoculations. This is in line with the close relatedness between this R. solanacearum phy II potato isolate and the R. solanacearum phy II isolates from rose, that was demonstrated in the phylogenetic analysis (Fig. 1 ). In previous studies, the R. solanacearum phy II potato isolate PD 2762 has been shown to be virulent on potato, tomato and eggplant as well (Overeem et al., 2023 ; Tjou-Tam-Sin et al., 2017a ). Thus, although the R. solanacearum phy II isolates from rose did not cause symptoms on rose, they can indeed cause severe damage in other solanaceous crops. The R. solanacearum phy II isolates from rose were not only able to cause disease symptoms on the above ground potato tissue, but they were able to spread to the newly formed daughter tubers as well (Fig. 3 ). Sometimes brown discoloration of the vascular bundle, i.e. typical brown rot symptoms, were present (Supplemental Fig. 2). Since tubers are used to vegetatively propagate potato plants, this could be a way of further spreading the disease. Especially in case of latently infected tubers, spreading of the disease to other areas could easily happen. For instance, Silveira et al. ( 2007 ) found that in some cases the daughter tubers of asymptomatic plants of different potato cultivars or clones tested positive for R . solanacearum with DAS-ELISA and/or PCR, while they were asymptomatic when they were cut open. Especially in countries with a poor seed potato production system and where seed tubers are only visually inspected instead of tested in the laboratory for latent infections, spread of R . solanacearum through latently infected tubers is a serious problem (Tessema et al., 2022 ). The origin of the R. solanacearum phy II isolates from rose and how they were introduced into the rose greenhouse cultivation remains unknown. Phylogenetic analysis showed that the R. solanacearum phy II isolates from rose were closely related to R. solanacearum phy II isolates from surface water in the Netherlands, isolated in 2010, 2018 and 2019 (Fig. 1 ). This close genetic relatedness could suggest that there might be a link between surface water and the infections in the rose greenhouse cultivation. R. solanacearum phy II infection of a crop as a result of contaminated surface water is a realistic scenario, that has happened in potato in the past. In 1995, R . solanacearum phy II infections were found on 94 Dutch farms (Schans & Steeghs, 1998 ). Some of the infected potato lots were clonally related to potato lots that had been irrigated with contaminated surface water. Infected potato lots on other farms did not have a clonal relationship with other potato lots, but were probably directly contaminated by using contaminated surface water for irrigation (Schans & Steeghs, 1998 ). Schans & Steeghs ( 1998 ) suggested that surface water in the Netherlands has been contaminated before 1992. They believed that irrigation of a potato lot with contaminated water in 1992 led to the infected offspring in 1995. Also in other countries R . solanacearum phy II has been found in surface water, for example in Germany (Retzer et al. 2006) and in England (Elphinstone et al. 1998). In England, two outbreaks of R . solanacearum phy II in potato have been related to irrigation with surface water from places where bittersweet infected with R . solanacearum phy II was growing. Because of the R . solanacearum phy II outbreaks related to contaminated surface water in the past, there has been an irrigation ban for seed potatoes in the Netherlands since 2005 (Janse, 2012 ). Furthermore, there are designated areas in which it is also prohibited to use surface water for irrigation of starch and ware potatoes and tomatoes. All three geographic locations where the R . solanacearum phy II isolates were found on rose, were located in close proximity to areas with an irrigation ban for starch and ware potatoes and tomatoes at the time of the findings in 2018. This makes surface water as a source of the R . solanacearum phy II infections in the rose greenhouse cultivation in 2018 likely. The opposite, i.e. R . solanacearum phy II infections in the rose greenhouse cultivation leading to contaminated surface water, cannot be excluded and should be taken into account. Although R. solanacearum phy II is already present in surface water in certain areas in the Netherlands, it could be introduced in waterways where it is not yet present, thereby forming a risk for crops grown near these waterways. Recently, indications for a similar occurrence for phylotype I were found (Vogelaar et al., 2023 ). R. pseudosolanacearum phy I, posing a potential threat to potato production (Overeem et al., 2023 ), has recently been found in surface water in the Netherlands at a restricted number of locations, and sequence analysis showed that the R. pseudosolanacearum phy I isolates found in surface water and bittersweet are closely related to the R. pseudosolanacearum phy I isolates that were found in rose before (Vogelaar et al., 2023 ), suggesting a potential link between the two. In conclusion, this study shows that the R. solanacearum phy II isolates from rose are phylogenetically related to the R. solanacearum phy II previously found in potato. Since the R. solanacearum phy II isolates from rose are highly virulent on potato, causing severe disease symptoms on both the above ground plant and the daughter tubers, spread to potato and other solanaceous crops is a serious risk for agriculture. Their unnoticed spread is a realistic scenario, since the bacteria were able to survive and multiply in rose plants without causing symptoms. This implies that rose can act as a reservoir for R . solanacearum phy II and, in this way, asymptomatic rose plants could potentially be involved in spreading the disease. Therefore, sampling and testing of asymptomatic rose plants and re-circulation water from the greenhouse rose cultivation remains a very important tool for restricting disease spread. Furthermore, this example of R. solanacearum phy II findings in the rose greenhouse cultivation stresses the importance of research focused on potential new host plants for this polyphagous plant pathogenic bacterium. Declarations The authors have no competing interests that are relevant to the content of this article to declare. Acknowledgements We would like to thank N.N.A. (Leon) Tjou-Tam-Sin for his valuable technical guidance with the inoculation experiments on rose. 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Supplementary Files SupplementalTable1.xlsx SupplementalTable2.xlsx SupplementalTable3.xlsx Supplementalfigures.pdf Cite Share Download PDF Status: Published Journal Publication published 08 Oct, 2024 Read the published version in European Journal of Plant Pathology → Version 1 posted Editorial decision: Minor revisions 15 Jul, 2024 Reviewers agreed at journal 02 Jun, 2024 Reviewers invited by journal 01 Jun, 2024 Editor invited by journal 24 May, 2024 Editor assigned by journal 21 May, 2024 First submitted to journal 19 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4396851","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":309503437,"identity":"bdf9d953-2fa9-414e-9c06-a0e5facac6dc","order_by":0,"name":"Nathalie Blom","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAr0lEQVRIiWNgGAWjYFACxgZmhgIGBn4oV4ZILQYMDJINEC4PUfaAtRgcIFaLOQNzA3OBgU208e0eM4kfDHcIa7FsADpshkFa7rY7Z8wkexieEdZicACohcfgcO62G2lpN3gYDpOgZfOMtLSbf0jSskEi+dht4mw5zNhwGOSXGTeSj/+WMSBGy/H2h48LKmxy+2ckNhu+qTgsR1ALMFIYDiCZQFjDKBgFo2AUjAIiAABMXDg/9jlBfwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0008-1408-6892","institution":"Netherlands Food and Consumer Product Safety Authority","correspondingAuthor":true,"prefix":"","firstName":"Nathalie","middleName":"","lastName":"Blom","suffix":""},{"id":309503438,"identity":"c9fff306-e8b6-4c61-bb39-eb319e9c0695","order_by":1,"name":"Peggy Gorkink-Smits","email":"","orcid":"","institution":"Netherlands Food and Consumer Product Safety Authority","correspondingAuthor":false,"prefix":"","firstName":"Peggy","middleName":"","lastName":"Gorkink-Smits","suffix":""},{"id":309503439,"identity":"d5de1eb5-297f-40d5-90bf-af7fa41bfc32","order_by":2,"name":"Marco Landman","email":"","orcid":"","institution":"Netherlands Food and Consumer Product Safety Authority","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Landman","suffix":""},{"id":309503440,"identity":"a72ec81d-25d5-49de-8329-0b1af3bc5111","order_by":3,"name":"Jeroen van de Bilt","email":"","orcid":"","institution":"Netherlands Food and Consumer Product Safety Authority","correspondingAuthor":false,"prefix":"","firstName":"Jeroen","middleName":"van","lastName":"de Bilt","suffix":""},{"id":309503441,"identity":"d1fd7a7f-1d67-4bcd-8274-4cb237cdfc73","order_by":4,"name":"Martijn Vogelaar","email":"","orcid":"","institution":"Netherlands Food and Consumer Product Safety Authority","correspondingAuthor":false,"prefix":"","firstName":"Martijn","middleName":"","lastName":"Vogelaar","suffix":""},{"id":309503442,"identity":"661e5529-4bff-4b5d-97d7-c0167ec82310","order_by":5,"name":"Tom Raaymakers","email":"","orcid":"","institution":"Netherlands Food and Consumer Product Safety Authority","correspondingAuthor":false,"prefix":"","firstName":"Tom","middleName":"","lastName":"Raaymakers","suffix":""},{"id":309503443,"identity":"7a0ed34d-4dfb-47ab-b026-2304e6a0d85d","order_by":6,"name":"Michael Visser","email":"","orcid":"","institution":"Netherlands Food and Consumer Product Safety Authority","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Visser","suffix":""},{"id":309503444,"identity":"bd00f0fe-97ef-4e5b-819d-1122927fcd8b","order_by":7,"name":"Michiel Pel","email":"","orcid":"","institution":"Netherlands Food and Consumer Product Safety Authority","correspondingAuthor":false,"prefix":"","firstName":"Michiel","middleName":"","lastName":"Pel","suffix":""},{"id":309503445,"identity":"1aa2b3b7-2cd9-402c-b575-26dd84e09c20","order_by":8,"name":"Maria Bergsma-Vlami","email":"","orcid":"","institution":"Netherlands Food and Consumer Product Safety Authority","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Bergsma-Vlami","suffix":""}],"badges":[],"createdAt":"2024-05-09 18:58:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4396851/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4396851/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10658-024-02960-8","type":"published","date":"2024-10-08T15:57:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58343773,"identity":"9dfca34d-2833-40ff-9ac4-d9cdcbe28fe6","added_by":"auto","created_at":"2024-06-14 07:21:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":289541,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic relatedness between \u003cem\u003eRalstonia\u003c/em\u003e isolates. (a) Phylogenetic tree based on average nucleotide identity (ANI) scores. Based on these scores, all phylotype II isolates from the Netherlands (1-8, 10-13) clustered together in one group. Scale bar represents a difference in ANI score of 0.8. (b) Minimum spanning tree showing the relatedness among \u003cem\u003eRalstonia solanacearum\u003c/em\u003e isolates originating from different host plants, water or soil from the Netherlands. Relatedness is based on a whole genome multilocus sequence typing (wgMLST) analysis. Edges are labeled with numbers representing the amount of genes with at least one single nucleotide polymorphism (SNP) between the two isolates connected by that edge\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4396851/v1/2b624bf0686906b496e8247e.png"},{"id":58343780,"identity":"5d174c68-f8f8-41fb-bb51-0959275bd564","added_by":"auto","created_at":"2024-06-14 07:21:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":998530,"visible":true,"origin":"","legend":"\u003cp\u003eDisease severity in potato. Disease class distribution per time point for each isolate in combination with potato cultivar “Saphir” (a) and cultivar “Talent” (c) at 20⁰C. Plants were scored on a scale from 0-3 where 0 = healthy plants, 1 = mild symptoms, 2 = severe symptoms and 3 = plant death. Area under de disease progress curve (AUDPC) for each isolate in combination with potato cultivar “Saphir” potato (b) and potato cultivar “Talent” (d). Error bars indicate standard error. Letters above the bars indicate significant differences (p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4396851/v1/82cba32f5ced4d0c34e95ac3.png"},{"id":58343774,"identity":"1f98f5f9-2b17-47b6-ba55-25fd667abeba","added_by":"auto","created_at":"2024-06-14 07:21:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":496798,"visible":true,"origin":"","legend":"\u003cp\u003eDaughter tuber formation and symptom development in daughter tubers after inoculation of potato plants. Number of daughter tubers per plant for potato cv. “Saphir” (a) at 41 dpi and potato cv. “Talent” (b) at 42 dpi, percentage of plants from which at least one daughter tuber was symptomatic for potato cv. “Saphir” (c) and potato cv. “Talent” (d), percentage of daughter tubers with symptoms for potato cv. “Saphir” (e) and potato cv. “Talent” (f), all at 20 °C. Error bars indicate standard error. Letters above the bars indicate significant differences (p\u0026lt;0.05). Dots in a and b represent the individual scores for each plant\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4396851/v1/39a3282a4173dc2e14e39e02.png"},{"id":66597067,"identity":"6461934e-b224-481e-8f81-0580a24aea49","added_by":"auto","created_at":"2024-10-14 16:06:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2137339,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4396851/v1/c8740267-36a5-4b3d-afa1-91160457786e.pdf"},{"id":58343778,"identity":"1b4d256b-7042-46e3-aced-33f4b5bb9714","added_by":"auto","created_at":"2024-06-14 07:21:11","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":12541,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4396851/v1/e4a01aee8491de07ce57e9fe.xlsx"},{"id":58345131,"identity":"08b6616e-0645-4090-be4d-28e8e42ade36","added_by":"auto","created_at":"2024-06-14 07:37:11","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":17847,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4396851/v1/247a12404ad643ad3bd0ba89.xlsx"},{"id":58344581,"identity":"a4a0667e-a925-4334-968d-4543768165f4","added_by":"auto","created_at":"2024-06-14 07:29:11","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":13029,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4396851/v1/24ac2734140da18eadff3a5c.xlsx"},{"id":58344584,"identity":"be089e2b-51d1-45be-b122-9ecdfed1cee7","added_by":"auto","created_at":"2024-06-14 07:29:11","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":402895,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4396851/v1/7d5300398ea5d2641526b493.pdf"}],"financialInterests":"","formattedTitle":"Ralstonia solanacearum (phylotype II) isolated from Rosa spp. in the Netherlands is closely related to phylotype II isolates from other sources in the Netherlands and is virulent on potato","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eRalstonia solanacearum\u003c/em\u003e is considered to be one of the most important plant pathogenic bacteria, due to its broad host range and the economic losses it causes, especially in potato (Mansfield et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In the past \u003cem\u003eR. solanacearum\u003c/em\u003e was considered a species complex (RSSC), composed of a heterogenous group of strains differing in host range and geographic origin (Mansfield et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and therefore, its members have been classified in several different ways. Examples are division into races (Buddenhagen \u0026amp; Kelman, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1964\u003c/span\u003e), into biotypes (Hayward, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1964\u003c/span\u003e) and more recently into four phylotypes, which are monophyletic clusters of strains that roughly correlate with the geographic origin of the isolates within these clusters (Prior \u0026amp; Fegan, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In 2014 the RSSC was split into three separate bacterial species based on gene sequences and DNA-DNA hybridization data. These species are \u003cem\u003eR. solanacearum\u003c/em\u003e (phy II), \u003cem\u003eRalstonia pseudosolanacearum\u003c/em\u003e (phy I and III) and \u003cem\u003eRalstonia syzygii\u003c/em\u003e (phy IV), of which the latter has been divided into three subspecies (Safni et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). All species and subspecies of the former species complex have a quarantine status within the European Union, except for \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esyzygii\u003c/em\u003e subsp. \u003cem\u003esyzygii\u003c/em\u003e (EU, 2019).\u003c/p\u003e \u003cp\u003eBacteria within the RSSC are soil-borne vascular pathogens. They colonize the xylem vessels of the plant, after which they spread systemically through their host. Accumulation of cells in the vascular tissue blocks the vessels and thereby impedes the water flow, leading to wilting symptoms (Lowe-Power et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Members of the RSSC are highly infectious and are able to survive in water (Alvarez et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Stevens et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Van Elsas et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Wenneker et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), soil (Van Elsas et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), and even on surfaces, including wood, rubber and metal (Wenneker et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). It is known that the weed bittersweet (\u003cem\u003eSolanum dulcamara\u003c/em\u003e) can get infected when it is in contact with contaminated surface water (Wenneker et al. 1999; Vogelaar et al., 2022). After infection, the bacteria can survive in these plants for years and can leak back into the water, allowing for recontamination of surface water after cold winters (Elphinstone et al. 1998). In addition to their broad host range, high infectiousness and ability to survive in different environments, another difficulty in preventing members of the RSSC to spread, is their frequent occurrence as latent infections. For example, \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e can be latently present on the surface, in the lenticels and in the vascular tissue of potato tubers (Sunaina et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), which poses a risk of spreading the disease without being noticed.\u003c/p\u003e \u003cp\u003eAn extensive outbreak of \u003cem\u003eR. solanacearum\u003c/em\u003e (phy II, race 3, biovar 2) in potato occurred in the Netherlands in 1995. In that year, 94 farms were found to be infected with the bacterium (Schans \u0026amp; Steeghs, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Most of these farms were either directly infected by using contaminated surface water for irrigation, or the infected lots were clonally propagated from seed potatoes of the cultivar \u0026lsquo;Bildtstar\u0026rsquo; that had been irrigated with infected surface water in previous years (Janse, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Schans \u0026amp; Steeghs, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). The control strategy focused on elimination of \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e from the entire potato production chain and prevention of new horizontal introductions into it (Schans \u0026amp; Steeghs, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). This approach has proven to be effective, in the fifteen years after the outbreak, the number of infected potato samples decreased substantially (Janse, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The current status of \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e in the potato production chain in the Netherlands is that it is transient, actionable with incidental findings (EPPO Reporting Service no. 01\u0026ndash;2017).\u003c/p\u003e \u003cp\u003eIn 2015, there has been a major outbreak of \u003cem\u003eR. pseudosolanacearum\u003c/em\u003e in \u003cem\u003eRosa\u003c/em\u003e spp. in the Netherlands. Greenhouse roses showed wilting symptoms, leaf chlorosis, early leaf drop, necrosis of pruned branches and in some cases bacterial slime protruding from the stem. Until then, rose was not known to be a host of any of the members of the RSSC (Tjou-Tam-Sin et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e). The causal agent was identified as \u003cem\u003eR\u003c/em\u003e. \u003cem\u003epseudosolanacearum\u003c/em\u003e phy I. Phylogenetic assignment based on amplified fragment length polymorphism data (AFLP) and multi locus phylogeny (MLSA) led to the conclusion that the \u003cem\u003eR. pseudosolanacearum\u003c/em\u003e phy I isolates from rose clustered together in one monophyletic group that contained, besides the isolates from rose, only one other isolate which originated from eggplant in India (Bergsma-Vlami et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Tjou-Tam-Sin et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e). This suggested a single and recent introduction of \u003cem\u003eR\u003c/em\u003e. \u003cem\u003epseudosolanacearum\u003c/em\u003e phy I into the rose industry in the Netherlands.\u003c/p\u003e \u003cp\u003eIn the years 2016\u0026ndash;2017 \u003cem\u003eR. pseudosolanacearum\u003c/em\u003e (phy I) was also found in greenhouse rose cultivation in other European countries (EPPO reporting service, articles 2016/039, 2017/018, 2017/085, and 2017/172). In 2017 similar symptoms as the ones in greenhouse roses in the Netherlands, were observed in cultivated roses in Korea (Kim et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Kim et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) were able to isolate \u003cem\u003eR. pseudosolanacearum\u003c/em\u003e (phy I) from rose plants from three different regions in Korea by plating and confirming the identity of suspect colonies with a phylotype-specific PCR.\u003c/p\u003e \u003cp\u003eFor a better understanding of the behavior of \u003cem\u003eR. pseudosolanacearum\u003c/em\u003e (phy I) on this new host plant, Tjou-Tam-Sin et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e) studied the effect of different factors on the virulence of these isolates on rose. They showed that rose cultivars differed in their susceptibility to \u003cem\u003eR\u003c/em\u003e. \u003cem\u003epseudosolanacearum\u003c/em\u003e (phy I) and that a higher temperature resulted in a higher disease incidence and disease severity (Tjou-Tam-Sin et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn 2018, during an annual survey in the Netherlands, \u003cem\u003eRalstonia\u003c/em\u003e sp. was found in rose plants again. This time \u003cem\u003eRalstonia\u003c/em\u003e sp. was isolated from asymptomatic plants. Immunofluorescence, real-time PCR, fatty acid analysis, sequence analysis (\u003cem\u003eegl\u003c/em\u003e and \u003cem\u003emut\u003c/em\u003eS), MALDI-TOF MS and a pathogenicity test were performed to identify the isolates. Remarkably, analysis of the \u003cem\u003eegl\u003c/em\u003e and \u003cem\u003emut\u003c/em\u003eS loci and MALDI-TOF MS revealed that five of these isolates, originating from three different geographic locations, did not belong to phylotype I, which had been consistently found in rose since 2015, but to phylotype II. This phylotype II has never been found in rose before and therefore there is currently no information available on the virulence of \u003cem\u003eR. solanacearum\u003c/em\u003e (phy II) on rose.\u003c/p\u003e \u003cp\u003eThe aim of this study was to determine the virulence of \u003cem\u003eR. solanacearum\u003c/em\u003e phy II, isolated from asymptomatic \u003cem\u003eRosa\u003c/em\u003e spp., on rose plants, under various conditions. Virulence was assessed using two different rose cultivars at two different temperatures. A whole genome multilocus sequence typing (wgMLST) analysis was additionally performed to determine the genetic relatedness of \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolated from symptomless \u003cem\u003eRosa\u003c/em\u003e spp. with \u003cem\u003eR. solanacearum\u003c/em\u003e (phy II) from potato, surface water, soil and other host plants previously found in the Netherlands. Furthermore, the virulence of the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II strains isolated from asymptomatic \u003cem\u003eRosa\u003c/em\u003e spp., was also assessed in potato plants, given the previous findings of \u003cem\u003eR. solanacearum\u003c/em\u003e phy II in seed potato and surface water in the Netherlands.\u003c/p\u003e"},{"header":"Materials \u0026 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial isolates\u003c/h2\u003e \u003cp\u003eTwo \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates, PD 7421 and PD 7394, that have been isolated from asymptomatic rose plants during an annual survey in 2018, were selected for inoculation of rose and potato plants (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In addition to these two isolates, an \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolate from potato was included in the inoculation experiments, as well as an \u003cem\u003eR\u003c/em\u003e. \u003cem\u003epseudosolanacearum\u003c/em\u003e phy I isolate from rose, which functioned as a positive control (PC; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Lyophilized bacterial cultures were transferred to nutrient agar (NA) broth and were placed in a shaker to incubate at room temperature for 0.5-1 hour. After incubation, the liquid medium was plated onto the general medium yeast peptone glucose agar (YPG). After two days, the bacterium was transferred to NA from a single colony. Two-day-old pure cultures were used to prepare the bacterial suspensions used in the inoculation experiments.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eRalstonia\u003c/em\u003e isolates used in the stem inoculation experiments on rose and potato plants.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExternal collection number(s)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eRalstonia\u003c/em\u003e species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePhylotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHost of origin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGeographic origin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYear of isolation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePD 2762\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eR. solanacearum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSolanum tuberosum\u003c/em\u003e cv. \u0026ldquo;Bildtstar\u0026rdquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eThe Netherlands\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1995\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePD 7394\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eR. solanacearum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eRosa\u003c/em\u003e sp. cv. \u0026ldquo;GL\u0026rdquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eThe Netherlands\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2018\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePD 7421\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eR. solanacearum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eRosa\u003c/em\u003e sp. cv. \u0026ldquo;Lucky Red\u0026rdquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eThe Netherlands\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2018\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePD 7123\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCFBP 8587\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eR. pseudosolanacearum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eRosa\u003c/em\u003e sp. cv. \u0026ldquo;Red Naomi\u0026rdquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eThe Netherlands\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2015\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eStem inoculation experiment in\u003c/b\u003e \u003cb\u003eRosa\u003c/b\u003e \u003cb\u003esp.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRose plants of cv. \u0026ldquo;Armando\u0026rdquo; and cv. \u0026ldquo;Red Naomi\u0026rdquo; were inoculated with a bacterial suspension (\u0026plusmn;\u0026thinsp;10^8 cfu/mL) or with sterile 0.01 M phosphate buffer (PB) as a negative control (NC). Inoculations were performed as described by Tjou-Tam-Sin et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e. Plants of cv. \u0026ldquo;Armando\u0026rdquo; were inoculated 3 weeks after pruning. New shoots were 0,5\u0026thinsp;\u0026minus;\u0026thinsp;1 cm thick at the moment of inoculation, and per plant 3 shoots were inoculated. Plants of cv. \u0026ldquo;Red Naomi\u0026rdquo; consisted of only one shoot, that was inoculated one week after pruning the flower. The experiment was conducted at a temperature of 20\u0026deg;C and 26\u0026deg;C, a relative humidity of 85% and a 14h/10h day/night cycle. For every combination of temperature and inoculum, 4 plants of each rose cultivar were inoculated.\u003c/p\u003e \u003cp\u003eDisease severity was scored on a scale from 0\u0026ndash;3 until 106 dpi, where 0\u0026thinsp;=\u0026thinsp;healthy plants, 1\u0026thinsp;=\u0026thinsp;mild symptoms (leaflets of compound leaves are hanging down and/or leaf veins start to yellow on one or a number of leaves, mainly at the bottom of the plant) 2\u0026thinsp;=\u0026thinsp;severe symptoms (expansion of yellowing leaf veins, chlorotic and necrotic leaves, early leaf drop) and 3\u0026thinsp;=\u0026thinsp;plant death, based on the scale described by Tjou-Tam-Sin et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e). Re-isolations were performed on all inoculated rose plants, either during the experiment or at the end of the experiment (at 106 dpi), in case of no symptom development. For re-isolations, plant extracts were made from pieces of petiole from the upper part of the plant (in case of symptomatic plants) or stem pieces from above the inoculation points of an inoculated stem (in case of asymptomatic plants). Symptomatic material was placed in 5 mL phosphate buffer saline (PBS) 0.01 M and incubated at room temperature for 30 minutes. After incubation, 50 \u0026micro;l extract was plated onto 6 SMSA plates by means of dilution plating. For asymptomatic plants, 15 stem pieces of \u0026plusmn;\u0026thinsp;1.5 cm of an inoculated branch were externally disinfected, crushed, and, after adding 7\u0026ndash;10 mL PB 0.05M, placed in a shaker for 30 minutes at 100 rpm at RT. Extract was centrifuged for 15 minutes at 7000 g. Supernatant was discarded and 1,5 mL PB 0.01M was added. Then, 50 \u0026micro;L was plated onto 6 SMSA plates by means of dilution plating. After 6 days of incubation at 28\u003csup\u003eo\u003c/sup\u003eC, plates were checked for typical colonies. Identity of these colonies was confirmed by MALDI-TOF MS (Van de Bilt et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003ePhylogenetic analysis was performed with the 23 isolates shown in Supplemental Table\u0026nbsp;1. Purified cultures were used to isolate DNA using the High Pure PCR Template Preparation Kit (Roche, according to the manufacturer\u0026rsquo;s instructions, except for the centrifugation step, which was performed at 6000 g instead of 8000 g. DNA sequencing was performed at GenomeScan (Leiden, the Netherlands) for Illumina 150PE (paired-end) sequencing using the NovaSeq 6000 with at least 2Gb output per sample. Samples were processed using the NEBNext\u0026reg; Ultra II FS DNA module and the NEBNext\u0026reg; Ultra II Ligation module. Fragmentation, A-tailing and ligation of sequencing adapters and PCR using NEBNext\u0026reg; Ultra II Q5 master mix of the resulting product was performed according to the procedure described in the NEBNext Ultra II FS DNA module and NEBNext Ultra II Ligation module instruction manual. After preparation, the quality and yield for all samples was measured with the Fragment Analyzer (Agilent Technologies, USA). The obtained read files, were deposited under BioProject accession PRJNA1089092. Illumina paired-end read quality was checked using fastqc (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.babraham.ac.uk/projects/fastqc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Subsequently, quality and adaptor trimming was performed using Trimmomatic v 0.39, with a sliding window of 5:30 and a minimum length at 50 nucleotides (Bolger et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Trimmed reads were input for a \u003cem\u003ede novo\u003c/em\u003e assembly using Spades v3.13.0 (Bankevich et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Prjibelski et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Assemblies were annotated using the NCBI prokaryotic genome annotation pipeline (PGAP version 6.5) (Tatusova et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and have been deposited at DDBJ/ENA/GenBank under the accessions indicated in Supplemental Table\u0026nbsp;1.Whole genome alignments were made with the Whole Genome Alignment plugin in CLC Genomics Workbench 21.0.5 (Qiagen, Aarhus, Denmark). Next, ANI comparisons were generated using the same plugin. From these ANI scores unweighted pair group method with arithmetic mean (UPGMA) trees were generated.\u003c/p\u003e \u003cp\u003eContig assemblies were utilized for a whole genome Multi Locus Sequence Typing (wgMLST) analysis using ChewBBACA v2.8.4 (Silva et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). To perform a wgMLST on the selected \u003cem\u003eRalstonia\u003c/em\u003e strains, first a training file was created for strain PD7642 using prodigal (Hyatt et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Subsequently, the training file and 106 \u003cem\u003eR. solanacearum\u003c/em\u003e genomes (Supplemental Table\u0026nbsp;2) were used to create a wgMLST scheme facilitated by the CreateSchema command in ChewBBACA. After creating the scheme, the wgMLST allele calling was performed for the 12 closely related isolates (Silva et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Visualization of the wgMLST results was performed by Phyloviz v2.0 (Nascimento et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn total 4343 loci were included in the wgMLST analysis. Each isolate was compared with all other isolates included in the analysis. For each combination of two isolates, the number of loci with \u0026gt;\u0026thinsp;1 SNP relative to each other was calculated and these numbers were used to create a minimum spanning tree. In the minimum spanning tree, the nodes (isolates) are connected by edges associated with numbers representing the number of loci with SNPs, in a way that every node is included and that the sum of the edges is as low as possible.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStem inoculation experiment in potato\u003c/h2\u003e \u003cp\u003ePotato plants of cv. \u0026ldquo;Saphir\u0026rdquo; were inoculated with a bacterial suspension (\u0026plusmn;\u0026thinsp;10^8 cfu/mL) or with 0.01 M PB as an NC. Plants were inoculated 18 days after planting the tubers, when plants were \u0026plusmn;\u0026thinsp;30 cm high. Per plant, 3 stems were inoculated at two different heights, just above and 10 cm above the soil level, using a syringe with a 0.5 x 16 mm needle. Per isolate 10 plants were inoculated. Plants were kept at 20\u0026deg;C (day and night) and a relative humidity of 60%. Disease severity was scored on a scale from 0\u0026ndash;3, for a period of 6 weeks. Plants were scored as 0 when there were no symptoms, as 1 when symptoms were only present on inoculated stems, as 2 when symptoms were also present on non-inoculated stems and as 3 when plants were dead. Furthermore, the total number of daughter tubers (\u0026gt;\u0026thinsp;1 cm) and the number of symptomatic daughter tubers per plant were counted. Re-isolations were performed on the above ground tissue of all plants at the end of the experiment, or during the experiment in case of heavily infected plants. Re-isolations were also performed on the newly formed tubers, as previously described (Overeem et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For this, a combined sample was taken per plant, containing pieces of the vascular bundle of each daughter tuber, regardless of symptom development. The experiment was repeated using the potato cultivar \u0026ldquo;Talent\u0026rdquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of re-isolates with MALDI-TOF MS\u003c/h2\u003e \u003cp\u003eColonies isolated from the inoculated rose and potato plants with a typical \u003cem\u003eR\u003c/em\u003e. (\u003cem\u003epseudo\u003c/em\u003e)\u003cem\u003esolanacearum\u003c/em\u003e morphology were analyzed on a Bruker MALDI Biotyper using the direct transfer method as described previously (Van de Bilt et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The obtained spectra were matched against the Bruker MSP library and against an \u003cem\u003ein-house\u003c/em\u003e created MSP library (Van de Bilt et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) that includes MSPs of all four RSSC phylotypes. Scores\u0026thinsp;\u0026gt;\u0026thinsp;2.00 are considered as a positive identification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using R (version 4.1.2) and the dunn. test package (version 1.3.5). The area under the disease progress curve (AUDPC) was calculated as described by Madden et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Averages of the AUDPC scores and percentage of daughter tubers with symptoms were compared using a Kruskal-Wallis rank sum test. Pairwise comparisons were performed using the Dunn\u0026rsquo;s test with a Hochberg correction of the p-value. For the number of daughter tubers an ANOVA followed by a Tukey multiple comparison of means test was performed.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eRalstonia solanacearum\u003c/b\u003e \u003cb\u003ephy II from rose causes latent infections in rose\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the virulence of \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolated from asymptomatic rose plants during the annual survey in 2018, isolates PD 7421 and PD 7394 were inoculated in rose plants of cultivars \u0026ldquo;Armando\u0026rdquo; and \u0026ldquo;Red Naomi\u0026rdquo; at two different temperatures. Both isolates did not cause any typical symptoms on rose regardless of the cultivar or temperature (Supplemental Fig.\u0026nbsp;1), resulting in an AUDPC of 0 after 106 days (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Also the potato \u003cem\u003eR. solanacearum\u003c/em\u003e (phy II) isolate PD 2762, did not cause any typical symptoms on rose in any of the treatments (Supplemental Fig.\u0026nbsp;1). In contrast, the \u003cem\u003eR. pseudosolanacearum\u003c/em\u003e phy I isolate PD7123, that is known to be highly virulent on rose, did cause symptoms (Supplemental Fig.\u0026nbsp;1) and resulted in an AUDPC ranging between 32.6\u0026ndash;91.9 depending on the rose cultivar and temperature (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Symptom development upon inoculation with PD7123 first started with leaflets of the compound leaves hanging down and/or yellowing of the veins in the leaves. After this, chlorosis expanded from the veins of the leaves to the whole leaf surface, and chlorotic leaves eventually turned necrotic. Sometimes, but not always, affected leaves fell off.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDisease severity, expressed as area under the disease progress curve (AUDPC), and proportion of highly infected plants of rose cultivars \u0026ldquo;Armando\u0026rdquo; and \u0026ldquo;Red Naomi\u0026rdquo; after stem inoculation with different \u003cem\u003eRalstonia\u003c/em\u003e isolates at 20\u0026deg;C and 26\u0026deg;C at 106 dpi.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRose cultivar\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAUDPC 20\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAUDPC 26\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProportion highly infected plants\u003csup\u003ea\u003c/sup\u003e 20\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eProportion highly infected plants\u003csup\u003ea\u003c/sup\u003e 26\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePD 7123\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArmando\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e73.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4/4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4/4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRed Naomi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e91.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4/4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4/4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePD 2762\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArmando\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3/4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3/4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRed Naomi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2/4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3/4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePD 7421\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArmando\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3/4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4/4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRed Naomi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2/4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePD 7394\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArmando\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3/4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2/4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRed Naomi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2/4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4/4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArmando\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0/4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRed Naomi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0/4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003ea\u003c/sup\u003ePlants from which\u0026thinsp;\u0026gt;\u0026thinsp;200 typical \u003cem\u003eRalstonia\u003c/em\u003e colonies were re-isolated.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003eb\u003c/sup\u003eLatent infection\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSince \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates PD 7421 and PD 7394 were isolated from asymptomatic plants, all inoculated rose plants were checked for the presence of these bacteria as latent infection, at the end of the experiment (106 dpi). In almost every treatment at least two of the four inoculated plants were found to be latently infected when re-isolations were performed, the only exception was \u0026ldquo;Red Naomi\u0026rdquo; inoculated with PD 7421 at 20\u0026deg;C (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). So, despite the lack of symptoms the pathogen is able to multiply inside the rose plants.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRalstonia solanacearum\u003c/b\u003e \u003cb\u003ephy II isolates from rose and other sources in the Netherlands show a close genetic relationship\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe genetic relatedness between the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates found in rose in 2018, PD 7421 and PD 7394, and 12 other \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates, originating from surface water, soil and different host plants, including potato, was investigated. For this, a whole genome alignment using 23 \u003cem\u003eRalstonia\u003c/em\u003e isolates (Supplemental Table\u0026nbsp;1) and pair-wise comparisons based on average nucleotide identity (ANI) scores were performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The majority of the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates included in the analysis clustered together in one monophyletic group with hardly any variation, this cluster contained only isolates originating from the Netherlands. Only two \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates, one from Ecuador (PD 3222-\u003cem\u003eAnthurium andreanum\u003c/em\u003e) and one from Brazil (PD 1414-\u003cem\u003eSolanum tuberosum\u003c/em\u003e), were placed outside this cluster and, with ANI scores of 97.6 and 96.4, respectively, were more distantly related to the 12 Dutch \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe dendrogram based on ANI values is not discriminative enough to show small genetic differences between the 12 \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates found in the Netherlands. Therefore, a whole genome multilocus sequence typing (wgMLST) analysis was performed and a minimum spanning tree was created (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The four \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from rose (numbers 1\u0026ndash;4) are very closely related to each other, and with a maximum number of genes with at least one SNP of 18 between number 2 (PD 7397) and number 3 (PD 7414, Supplemental Table\u0026nbsp;3), they might be considered clonally related. The isolates from rose are also very closely related to the three \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from surface water in the Netherlands (numbers 5\u0026ndash;7) and to \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolate PD 2762 (number 13), which was isolated from potato in 1995. The number of genes with at least one SNP between a \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolate from rose and an \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolate from surface water ranged between 41 and 60 (Supplemental Table\u0026nbsp;3). Within the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II surface water isolates group, this number ranged from 22 to 34. Between the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II rose isolates and the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II potato isolate, this number ranged from 86 to 91. With a number of genes with at least one SNP ranging from 250 to 501, the group of \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates on the top left (numbers 8 and 10\u0026ndash;12), isolated from different crops and soil, are more distantly related to the four \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from rose (Supplemental Table\u0026nbsp;3), although they can still be seen as closely related isolates. Together these results show that the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from rose are very closely related to the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates that have been present in the Netherlands for years, if not decades.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRalstonia solanacearum\u003c/b\u003e \u003cb\u003ephy II isolates from rose are virulent on potato\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo test the virulence of the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from rose on potato, two potato cultivars, \u0026ldquo;Saphir\u0026rdquo; and \u0026ldquo;Talent\u0026rdquo;, were inoculated with isolates PD 7394 and PD 7421 at a temperature resembling the temperate climate (20\u0026deg;C). Additionally, plants were inoculated with \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolate PD 2762 which is known to be highly virulent on potato and the \u003cem\u003eR\u003c/em\u003e. \u003cem\u003epseudosolanacearum\u003c/em\u003e phy I isolate PD 7123 from rose (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) which has been shown to be less virulent on potato at lower temperatures. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea shows symptom development in the above ground potato tissue of cv. \u0026ldquo;Saphir\u0026rdquo; over a period of 41 days. Cv. \u0026ldquo;Saphir\u0026rdquo; plants inoculated with \u003cem\u003eR. solanacearum\u003c/em\u003e phy II, showed the first symptoms at 8 dpi. In plants inoculated with \u003cem\u003eR\u003c/em\u003e. \u003cem\u003epseudosolanacearum\u003c/em\u003e phy I, the first symptoms appeared several days later, at 11 dpi. From 11 dpi onwards, plants inoculated with \u003cem\u003eR. solanacearum\u003c/em\u003e phy II started to show severe disease symptoms, as wilting was visible in the non-inoculated stems. At the end of the experiment, at 41 dpi, all cv. \u0026ldquo;Saphir\u0026rdquo; potato plants inoculated with either \u003cem\u003eR. solanacearum\u003c/em\u003e phy II from potato, or \u003cem\u003eR. solanacearum\u003c/em\u003e phy II from rose, showed severe disease symptoms or had died. In contrast, cv. \u0026ldquo;Saphir\u0026rdquo; plants inoculated with \u003cem\u003eR\u003c/em\u003e. \u003cem\u003epseudosolanacearum\u003c/em\u003e phy I had only developed mild symptoms or no symptoms at all at 20\u0026deg;C at 41 dpi. The AUDPC for cv. \u0026ldquo;Saphir\u0026rdquo; plants inoculated with any of the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates was significantly higher than for plants inoculated with \u003cem\u003eR\u003c/em\u003e. \u003cem\u003epseudosolanacearum\u003c/em\u003e phy I or the NC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). However, no significant differences were found in the AUDPC between cv. \u0026ldquo;Saphir\u0026rdquo; plants inoculated with \u003cem\u003eR. solanacearum\u003c/em\u003e phy II from rose and plants inoculated with \u003cem\u003eR. solanacearum\u003c/em\u003e phy II from potato (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen the experiment was repeated with cv. \u0026ldquo;Talent\u0026rdquo;, in all treatments, including \u003cem\u003eR\u003c/em\u003e. \u003cem\u003epseudosolanacearum\u003c/em\u003e phy I, the first mild symptoms appeared at 8 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). After 10 days, also the non-inoculated stems started to wilt in cv. \u0026ldquo;Talent\u0026rdquo; plants inoculated with any of the \u003cem\u003eR. solanacearum\u003c/em\u003e phylotype II isolates. For \u003cem\u003eR\u003c/em\u003e. \u003cem\u003epseudosolanacearum\u003c/em\u003e phy I, severe symptoms did not appear before day 15. Similarly to cv. \u0026ldquo;Saphir\u0026rdquo;, all cv. \u0026ldquo;Talent\u0026rdquo; plants inoculated with either \u003cem\u003eR. solanacearum\u003c/em\u003e phy II from potato, or \u003cem\u003eR. solanacearum\u003c/em\u003e phy II from rose showed severe symptoms or had died at 42 dpi. Plants of cv. \u0026ldquo;Talent\u0026rdquo; inoculated with \u003cem\u003eR\u003c/em\u003e. \u003cem\u003epseudosolanacearum\u003c/em\u003e phy I did not reach the stage of plant death, however all plants did develop some symptoms (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Similar to cv. \u0026ldquo;Saphir\u0026rdquo; also for cv. \u0026ldquo;Talent\u0026rdquo; the AUDPC was higher for the three \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates than for the \u003cem\u003eR\u003c/em\u003e. \u003cem\u003epseudosolanacearum\u003c/em\u003e phy I isolate or the NC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). However, while the AUDPC for the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates PD 2762 (potato) and PD 7394 (rose) were significantly higher than the AUDPC for the \u003cem\u003eR\u003c/em\u003e. \u003cem\u003epseudosolanacearum\u003c/em\u003e phy I PD 7123 (rose) and the NC, due to higher variability the difference between the AUDPC for the rose isolates PD 7421 (\u003cem\u003eR. solanacearum\u003c/em\u003e phy II) and PD 7123 (\u003cem\u003eR\u003c/em\u003e. \u003cem\u003epseudosolanacearum\u003c/em\u003e phy I) was not significant. Again, no significant difference was found in the AUDPC between the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from rose and the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolate from potato (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003cb\u003eInoculation with\u003c/b\u003e \u003cb\u003eRalstonia solanacearum\u003c/b\u003e \u003cb\u003ephy II isolates from rose leads to decreased daughter tuber formation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo test the effect of inoculation with \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from rose on tuber formation in potato, the number of daughter tubers formed were counted per potato plant. Figures\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb show the number of daughter tubers formed at 20\u0026deg;C for plants of cv. \u0026ldquo;Saphir\u0026rdquo; and cv. \u0026ldquo;Talent\u0026rdquo; inoculated with the different bacterial isolates at 41 and 42 dpi, respectively. All \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates, from both rose and potato, greatly reduced the number of daughter tubers formed compared to the NC. In contrast, this reduction in daughter tuber formation was not seen for \u003cem\u003eR. pseudosolanacearum\u003c/em\u003e phy I. This result was valid for both cv. \u0026ldquo;Saphir\u0026rdquo; and cv. \u0026ldquo;Talent\u0026rdquo;.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eInoculation of potato plants with\u003c/b\u003e \u003cb\u003eRalstonia solanacearum\u003c/b\u003e \u003cb\u003ephy II isolates from rose leads to symptom development in daughter tubers\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNot only the number of daughter tubers formed, but also the number of symptomatic daughter tubers present in potato plants inoculated with \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates, from both rose and potato, or with \u003cem\u003eR. pseudosolanacearum\u003c/em\u003e phy I from rose was counted per individual potato plant. Plants of cv. \u0026ldquo;Saphir\u0026rdquo; inoculated with \u003cem\u003eR. solanacearum\u003c/em\u003e phy II from rose either formed no daughter tubers at all, or formed tubers of which at least one daughter tuber showed disease symptoms, irrespective of the rose \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolate used. The same was valid for cv. \u0026ldquo;Saphir\u0026rdquo; plants inoculated with the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolate from potato (PD 2762). In contrast, only one plant of the cv. \u0026ldquo;Saphir\u0026rdquo; inoculated with \u003cem\u003eR. pseudosolanacearum\u003c/em\u003e phy I from rose formed at least one daughter tuber with symptoms, while all other cv. \u0026ldquo;Saphir\u0026rdquo; plants only formed asymptomatic daughter tubers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Also the percentage of symptomatic daughter tubers was higher for cv. \u0026ldquo;Saphir\u0026rdquo; plants inoculated with \u003cem\u003eR. solanacearum\u003c/em\u003e phy II, from either rose or potato, than for cv. \u0026ldquo;Saphir\u0026rdquo; plants inoculated with \u003cem\u003eR. pseudosolanacearum\u003c/em\u003e phy I (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eAll plants of cv. \u0026ldquo;Talent\u0026rdquo; inoculated with \u003cem\u003eR. solanacearum\u003c/em\u003e phy II from potato formed at least one symptomatic daughter tuber, in case daughter tubers were formed at all. Also plants of cv. \u0026ldquo;Talent\u0026rdquo; that were inoculated with one of the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from rose and formed daughter tubers, formed at least one symptomatic daughter tuber, with the exception of one plant inoculated with PD 7421, which only formed asymptomatic daughter tubers. In contrast with cv. \u0026ldquo;Saphir\u0026rdquo;, all cv. \u0026ldquo;Talent\u0026rdquo; plants inoculated with \u003cem\u003eR. pseudosolanacearum\u003c/em\u003e phy I formed at least 1 symptomatic daughter tuber (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). The mean percentage of symptomatic daughter tubers, however, appears lower for plants inoculated with \u003cem\u003eR. pseudosolanacearum\u003c/em\u003e phy I than for plants inoculated with any of the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates, although this trend was found not to represent a significant difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSince the outbreak of \u003cem\u003eR\u003c/em\u003e. \u003cem\u003epseudosolanacearum\u003c/em\u003e phy I in rose in 2015, annual surveys have been conducted in the greenhouse cultivation of rose in the Netherlands. The finding of the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates in asymptomatic rose plants in 2018, however, was unexpected, because until then the \u003cem\u003eRalstonia\u003c/em\u003e isolates found in rose always belonged to \u003cem\u003eR. pseudosolanacearum\u003c/em\u003e phy I (Bergsma-Vlami et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Tjou-Tam-Sin et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e). Although \u003cem\u003eR. solanacearum\u003c/em\u003e phy II was known to cause disease in other crops, for example brown rot in potato, rose was not known to be a natural host of \u003cem\u003eR. solanacearum\u003c/em\u003e phy II at that time.\u003c/p\u003e \u003cp\u003eStem inoculation of the rose cultivars \u0026ldquo;Armando\u0026rdquo; and \u0026ldquo;Red Naomi\u0026rdquo; with two of the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from rose, PD 7394 and PD 7421, did not lead to symptom development on these rose plants at 20\u0026deg;C, nor at 26\u0026deg;C (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This lack of symptom development is in line with the origin of the isolates, i.e. asymptomatic rose plants. Only two rose \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates were inoculated in the rose plants, but similar results are expected for the other \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from the findings in rose in 2018 (Supplemental Table\u0026nbsp;1), considering their great genetic similarity, as confirmed by the phylogenetic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). How rose plants respond to inoculation with more distantly related \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates, for instance the isolates originating from Ecuador and Brazil (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), remains unknown.\u003c/p\u003e \u003cp\u003eAlthough \u003cem\u003eR. solanacearum\u003c/em\u003e phy II PD 7394 and PD 7421 were not able to cause disease symptoms on the rose plants, re-isolations from inoculated rose plants resulted in high numbers of typical \u003cem\u003eRalstonia\u003c/em\u003e colonies in almost every treatment (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This indicates that these \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates are able to survive and multiply inside the rose plants. Latent infections of \u003cem\u003eR. solanacearum\u003c/em\u003e phy II do occur in other plant species as well, including potato (Priou et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Silveira et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Sunaina et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), tomato (Grimault \u0026amp; Prior, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Gutarra et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), pepper (Grimault \u0026amp; Prior, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), eggplant (Grimault \u0026amp; Prior, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Gutarra et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and geranium (Swanson et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Swanson et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Upon inoculation in rose, latent infections with \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e phy II originating from plant species other than rose have been reported before by Tjou-Tam-Sin et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e). Similar to our results, inoculation with all \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates led to a disease incidence of 0%, but in all cases latent infections were confirmed (Tjou-Tam-Sin et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e). Latent infections in rose might not lead to visible damage to the rose plants themselves, and thus do not cause any obvious economic damage. However, these infections do form a risk for other crops, as unseen spread of the bacteria to other areas and crops can easily occur. In the past, spread of \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e phy II through latently infected plant material occurred, for instance, when geranium cuttings were imported from Kenya, Guatemala and Costa Rica into the US (Swanson et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). A similar event occurred in Europe when \u003cem\u003ePelargonium\u003c/em\u003e cuttings from Kenya, that were infected with \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e phy II, were imported into European countries (Janse et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince the Netherlands is the biggest producer of certified seed potato in Northwestern Europe (Goffart et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and there has been an extensive outbreak of \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e phy II in potato in the past (Schans \u0026amp; Steeghs, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), it was highly relevant to test how potato plants respond to inoculation with the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from rose. In contrast to what was seen in the inoculation experiment on rose, the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from rose were highly virulent on potato plants at 20\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Actually, the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from rose were just as harmful on potato as the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolate, PD 2762, isolated from potato in the Netherlands in 1995, that was included as a positive control (PC) for the inoculations. This is in line with the close relatedness between this \u003cem\u003eR. solanacearum\u003c/em\u003e phy II potato isolate and the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from rose, that was demonstrated in the phylogenetic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In previous studies, the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II potato isolate PD 2762 has been shown to be virulent on potato, tomato and eggplant as well (Overeem et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Tjou-Tam-Sin et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e). Thus, although the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from rose did not cause symptoms on rose, they can indeed cause severe damage in other solanaceous crops.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from rose were not only able to cause disease symptoms on the above ground potato tissue, but they were able to spread to the newly formed daughter tubers as well (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Sometimes brown discoloration of the vascular bundle, i.e. typical brown rot symptoms, were present (Supplemental Fig.\u0026nbsp;2). Since tubers are used to vegetatively propagate potato plants, this could be a way of further spreading the disease. Especially in case of latently infected tubers, spreading of the disease to other areas could easily happen. For instance, Silveira et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) found that in some cases the daughter tubers of asymptomatic plants of different potato cultivars or clones tested positive for \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e with DAS-ELISA and/or PCR, while they were asymptomatic when they were cut open. Especially in countries with a poor seed potato production system and where seed tubers are only visually inspected instead of tested in the laboratory for latent infections, spread of \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e through latently infected tubers is a serious problem (Tessema et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe origin of the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from rose and how they were introduced into the rose greenhouse cultivation remains unknown. Phylogenetic analysis showed that the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from rose were closely related to \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from surface water in the Netherlands, isolated in 2010, 2018 and 2019 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This close genetic relatedness could suggest that there might be a link between surface water and the infections in the rose greenhouse cultivation. \u003cem\u003eR. solanacearum\u003c/em\u003e phy II infection of a crop as a result of contaminated surface water is a realistic scenario, that has happened in potato in the past. In 1995, \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e phy II infections were found on 94 Dutch farms (Schans \u0026amp; Steeghs, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Some of the infected potato lots were clonally related to potato lots that had been irrigated with contaminated surface water. Infected potato lots on other farms did not have a clonal relationship with other potato lots, but were probably directly contaminated by using contaminated surface water for irrigation (Schans \u0026amp; Steeghs, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Schans \u0026amp; Steeghs (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) suggested that surface water in the Netherlands has been contaminated before 1992. They believed that irrigation of a potato lot with contaminated water in 1992 led to the infected offspring in 1995. Also in other countries \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e phy II has been found in surface water, for example in Germany (Retzer et al. 2006) and in England (Elphinstone et al. 1998). In England, two outbreaks of \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e phy II in potato have been related to irrigation with surface water from places where bittersweet infected with \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e phy II was growing.\u003c/p\u003e \u003cp\u003eBecause of the \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e phy II outbreaks related to contaminated surface water in the past, there has been an irrigation ban for seed potatoes in the Netherlands since 2005 (Janse, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Furthermore, there are designated areas in which it is also prohibited to use surface water for irrigation of starch and ware potatoes and tomatoes. All three geographic locations where the \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e phy II isolates were found on rose, were located in close proximity to areas with an irrigation ban for starch and ware potatoes and tomatoes at the time of the findings in 2018. This makes surface water as a source of the \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e phy II infections in the rose greenhouse cultivation in 2018 likely.\u003c/p\u003e \u003cp\u003eThe opposite, i.e. \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e phy II infections in the rose greenhouse cultivation leading to contaminated surface water, cannot be excluded and should be taken into account. Although \u003cem\u003eR. solanacearum\u003c/em\u003e phy II is already present in surface water in certain areas in the Netherlands, it could be introduced in waterways where it is not yet present, thereby forming a risk for crops grown near these waterways. Recently, indications for a similar occurrence for phylotype I were found (Vogelaar et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cem\u003eR. pseudosolanacearum\u003c/em\u003e phy I, posing a potential threat to potato production (Overeem et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), has recently been found in surface water in the Netherlands at a restricted number of locations, and sequence analysis showed that the \u003cem\u003eR. pseudosolanacearum\u003c/em\u003e phy I isolates found in surface water and bittersweet are closely related to the \u003cem\u003eR. pseudosolanacearum\u003c/em\u003e phy I isolates that were found in rose before (Vogelaar et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), suggesting a potential link between the two.\u003c/p\u003e \u003cp\u003eIn conclusion, this study shows that the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from rose are phylogenetically related to the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II previously found in potato. Since the \u003cem\u003eR. solanacearum\u003c/em\u003e phy II isolates from rose are highly virulent on potato, causing severe disease symptoms on both the above ground plant and the daughter tubers, spread to potato and other solanaceous crops is a serious risk for agriculture. Their unnoticed spread is a realistic scenario, since the bacteria were able to survive and multiply in rose plants without causing symptoms. This implies that rose can act as a reservoir for \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e phy II and, in this way, asymptomatic rose plants could potentially be involved in spreading the disease. Therefore, sampling and testing of asymptomatic rose plants and re-circulation water from the greenhouse rose cultivation remains a very important tool for restricting disease spread. Furthermore, this example of \u003cem\u003eR. solanacearum\u003c/em\u003e phy II findings in the rose greenhouse cultivation stresses the importance of research focused on potential new host plants for this polyphagous plant pathogenic bacterium.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors have no competing interests that are relevant to the content of this article to declare.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe would like to thank N.N.A. (Leon) Tjou-Tam-Sin for his valuable technical guidance with the inoculation experiments on rose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlvarez, B., L\u0026oacute;pez, M. M., \u0026amp; Biosca, E. G. (2008). 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(2001).\u0026nbsp;Assessment of latent infection frequency in progeny tubers of advanced potatoclones resistant to bacterial wilt: A new selection criterion. \u003cem\u003ePotato research,\u003c/em\u003e \u003cem\u003e44\u003c/em\u003e(4), 359-373.\u003c/li\u003e\n \u003cli\u003ePrjibelski, A., Antipov, D., Meleshko, D., Lapidus, A., \u0026amp; Korobeynikov, A. (2020). Using SPAdes de novo assembler. \u003cem\u003eCurrent protocols in bioinformatics,\u003c/em\u003e \u003cem\u003e70\u003c/em\u003e(1), e102.\u003c/li\u003e\n \u003cli\u003eSafni, I., Cleenwerck, I., De Vos, P., Fegan, M., Sly, L., \u0026amp; Kappler, U. (2014).\u0026nbsp;Polyphasic taxonomic revision of the Ralstonia solanacearum species complex: proposal to emend the descriptions of Ralstonia solanacearum and Ralstonia syzygii and reclassify current R. syzygii strains as Ralstonia syzygii subsp. syzygii subsp. nov., R. s. \u003cem\u003eInternational journal of systematic and evolutionary microbiology,\u003c/em\u003e \u003cem\u003e64\u003c/em\u003e(Pt_9), 3087-3103.\u003c/li\u003e\n \u003cli\u003eSchans, J., \u0026amp; Steeghs, M. (1998). Strategy and results of eradication of brown rot in The Netherlands 1. \u003cem\u003eEPPO Bulletin,\u003c/em\u003e \u003cem\u003e28\u003c/em\u003e(1‐2), 121-133.\u003c/li\u003e\n \u003cli\u003eSilva, M., Machado, M. P., Silva, D. N., Rossi, M., Moran-Gilad, J., Santos, S., Ramirez, M., \u0026amp; Carrico, J. A. (2018). chewBBACA: A complete suite for gene-by-gene schema creation and strain identification. \u003cem\u003eMicrobial genomics,\u003c/em\u003e \u003cem\u003e4\u003c/em\u003e(3), e000166.\u003c/li\u003e\n \u003cli\u003eSilveira, J. R., Duarte, V., Moraes, M. G., Lopes, C. A., Fernandes, J. M., Barni, V., \u0026amp; Maciel, J. L. (2007). Epidemiological analysis of clones and cultivars of potato in soil naturally infested with Ralstonia solanacearum biovar 2. \u003cem\u003eFitopatologia Brasileira,\u003c/em\u003e \u003cem\u003e32\u003c/em\u003e(3), 181-188.\u003c/li\u003e\n \u003cli\u003eStevens, L. H., Van der Zouwen, P. S., Van Tongeren, C. A. M., Kastelein, P., \u0026amp; Van der Wolf, J. M. (2018).\u0026nbsp;Survival of Ralstonia solanacearum and Ralstonia pseudosolanacearum in drain water. \u003cem\u003eEPPO Bulletin,\u003c/em\u003e \u003cem\u003e48\u003c/em\u003e(1), 97-104.\u003c/li\u003e\n \u003cli\u003eSunaina, V., Kishore, V., \u0026amp; Shekhawat, G. (1989). Latent survival of Pseudomonas solanacearum in potato tubers and weeds/Latentes \u0026Uuml;berleben von Pseudomonas solanacearum in Kartoffelknollen und Unkr\u0026auml;utern. \u003cem\u003eZeitschrift f\u0026uuml;r Pflanzenkrankheiten und Pflanzenschutz/Journal of Plant Diseases and Protection,\u003c/em\u003e \u003cem\u003e96\u003c/em\u003e(4), 361-364.\u003c/li\u003e\n \u003cli\u003eSwanson, J. K., Yao, J., Tans-Kersten, J., \u0026amp; Allen, C. (2005). Behavior of Ralstonia solanacearum race 3 biovar 2 during latent and active infection of geranium. \u003cem\u003ePhytopathology,\u003c/em\u003e \u003cem\u003e95\u003c/em\u003e(2), 136-143.\u003c/li\u003e\n \u003cli\u003eSwanson, J. K., Montes, L., Mejia, L., \u0026amp; Allen, C. (2007). Detection of latent infections of Ralstonia solanacearum race 3 biovar 2 in geranium. \u003cem\u003ePlant disease,\u003c/em\u003e \u003cem\u003e91\u003c/em\u003e(7), 828-834.\u003c/li\u003e\n \u003cli\u003eTatusova, T., DiCuccio, M., Badretdin, A., Chetvernin, V., Nawrocki, E. P., Zaslavsky, L., Lomsadze, A., Pruitt, K. D., Borodovsky, M., \u0026amp; Ostell, J. (2016). NCBI prokaryotic genome annotation pipeline. \u003cem\u003eNucleic acids research,\u003c/em\u003e \u003cem\u003e44\u003c/em\u003e(14), 6614-6624.\u003c/li\u003e\n \u003cli\u003eTessema, L., Seid, E., W/Giorgis, G., Sharma, K., Workie, M., Negash, K., Misganaw, A., \u0026amp; Abebe, T. (2022). Incidence and Occurrence of Latent Ralstonia solanacearum Infection in Seed Potato from Farmer Seed Grower Cooperatives in Southern and Central Ethiopia. \u003cem\u003ePotato research,\u003c/em\u003e \u003cem\u003e65\u003c/em\u003e(3), 649-662.\u003c/li\u003e\n \u003cli\u003eTjou-Tam-Sin, N. N. A., Van de Bilt, J. L. J., Westenberg, M., Gorkink-Smits, P. P. M. A., Landman, N. M., \u0026amp; Bergsma-Vlami, M. (2017a). Assessing the pathogenic ability of Ralstonia pseudosolanacearum (Ralstonia solanacearum phylotype I) from ornamental Rosa spp. plants. \u003cem\u003eFrontiers in plant science,\u003c/em\u003e \u003cem\u003e8\u003c/em\u003e, 1895.\u003c/li\u003e\n \u003cli\u003eTjou-Tam-Sin, N. N. A., Van de Bilt, J. L. J., Westenberg, M., Bergsma-Vlami, M., Korpershoek, H. J., Vermunt, A. M. W., Meekes, E. T. M., Teunissen, H. A. S., \u0026amp; Van Vaerenbergh, J. (2017b).\u0026nbsp;First report of bacterial wilt caused by Ralstonia solanacearum in ornamental Rosa sp. \u003cem\u003ePlant disease,\u003c/em\u003e \u003cem\u003e101\u003c/em\u003e(2), 378-378.\u003c/li\u003e\n \u003cli\u003eVan de Bilt, J., Wolsink, M., Gorkink-Smits, P., Landman, N., \u0026amp; Bergsma-Vlami, M. (2018). Application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry for rapid and accurate identification of Ralstonia solanacearum and Ralstonia pseudosolanacearum. \u003cem\u003eEuropean Journal of Plant Pathology,\u003c/em\u003e \u003cem\u003e152\u003c/em\u003e(4), 921-931.\u003c/li\u003e\n \u003cli\u003eVan Elsas, J. D., Kastelein, P., De Vries, P. M., \u0026amp; Van Overbeek, L. S. (2001).\u0026nbsp;Effects of ecological factors on the survival and physiology of Ralstonia solanacearum bv. 2 in irrigation water. \u003cem\u003eCanadian Journal of Microbiology,\u003c/em\u003e \u003cem\u003e47\u003c/em\u003e(9), 842-854.\u003c/li\u003e\n \u003cli\u003eVan Elsas, J. D., Kastelein, P., Van Bekkum, P., Van der Wolf, J. M., De Vries, P. M., \u0026amp; Van Overbeek, L. S. (2000).\u0026nbsp;Survival of Ralstonia solanacearum biovar 2, the causative agent of potato brown rot, in field and microcosm soils in temperate climates. \u003cem\u003ePhytopathology,\u003c/em\u003e \u003cem\u003e90\u003c/em\u003e(12), 1358-1366.\u003c/li\u003e\n \u003cli\u003eVogelaar, M., Van de Bilt, J., Blom, N., Pel, C., Van Doorn, B., Landman, M., Gorkink, P., Raaymakers, T., Vreeburg, R., \u0026amp; Bergsma-Vlami, M. (2023).\u0026nbsp;Presence of Ralstonia pseudosolanacearum (phylotype I) in aquatic environments in the Netherlands. \u003cem\u003ePlant Disease,\u003c/em\u003e \u003cem\u003e107\u003c/em\u003e(8), 2320-2324.\u003c/li\u003e\n \u003cli\u003eWenneker, M., Van Beuningen, A., Van Nieuwenhuijze, A., \u0026amp; Janse, J. (1998).\u0026nbsp;Survival of brown rot and disinfection of surface water. The survival of the brown rot bacteria (Pseudomonas solanacearum) in several substrates and the efficiency of several methods for the disinfection of surface water. \u003cem\u003eGewasbescherming,\u003c/em\u003e \u003cem\u003e29\u003c/em\u003e(1), 7-11.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Ralstonia solanacearum, phylotype II, Rosa spp., Solanum tuberosum, virulence, phylogenetic analysis","lastPublishedDoi":"10.21203/rs.3.rs-4396851/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4396851/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn 2018, during an annual survey in the Netherlands, \u003cem\u003eRalstonia solanacearum\u003c/em\u003e phylotype II (phy II) was found in asymptomatic greenhouse rose plants at three geographic locations. These findings were remarkable, since previous findings of \u003cem\u003eRalstonia\u003c/em\u003e sp. in rose always concerned \u003cem\u003eRalstonia pseudosolanacearum\u003c/em\u003e phylotype I (phy I). Therefore, no information was available on the virulence of \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e phy II on rose. In this study, \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e phy II isolates PD 7421 and PD 7394, isolated in 2018 from asymptomatic ornamental rose (\u003cem\u003eRosa\u003c/em\u003e spp.), were assessed for their virulence in two rose cultivars (\u0026ldquo;Armando\u0026rdquo; and \u0026ldquo;Red Naomi\u0026rdquo;) at two temperatures. No typical symptoms were observed for PD 7421 and PD 7394 on the two rose cultivars, irrespective of the temperature. However, latent infections upon inoculation of these isolates on rose did occur. \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e phy II is known as a major potato pathogen, where it causes brown rot. Whole genome multilocus sequence typing analysis demonstrated that the \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e phy II isolates from rose were closely related to \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e phy II isolates previously found in seed potato and surface water in the Netherlands. Because of this close genetic relatedness, the virulence of PD 7421 and PD 7394 was also assessed in potato plants, where both isolates caused severe disease symptoms on the shoots as well as the daughter tubers. This implies that rose can act as a reservoir for \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e phy II and, in this way, can potentially be involved in spreading this bacterium.\u003c/p\u003e","manuscriptTitle":"Ralstonia solanacearum (phylotype II) isolated from Rosa spp. in the Netherlands is closely related to phylotype II isolates from other sources in the Netherlands and is virulent on potato","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-14 07:21:06","doi":"10.21203/rs.3.rs-4396851/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2024-07-15T15:04:15+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-06-02T05:50:32+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-02T02:21:16+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"European Journal of Plant Pathology","date":"2024-05-24T04:03:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-21T10:57:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Plant Pathology","date":"2024-05-19T09:11:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2840d940-6e30-42d5-8414-208faa27dd32","owner":[],"postedDate":"June 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-14T15:59:37+00:00","versionOfRecord":{"articleIdentity":"rs-4396851","link":"https://doi.org/10.1007/s10658-024-02960-8","journal":{"identity":"european-journal-of-plant-pathology","isVorOnly":false,"title":"European Journal of Plant Pathology"},"publishedOn":"2024-10-08 15:57:06","publishedOnDateReadable":"October 8th, 2024"},"versionCreatedAt":"2024-06-14 07:21:06","video":"","vorDoi":"10.1007/s10658-024-02960-8","vorDoiUrl":"https://doi.org/10.1007/s10658-024-02960-8","workflowStages":[]},"version":"v1","identity":"rs-4396851","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4396851","identity":"rs-4396851","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}