Investigation of potassium tetraborate resistance in Dickeya spp. Honors Thesis Presented to the College of Arts and Sciences, Cornell University in Partial Fulfillment of the Requirements for the Biological Sciences Honors Program by Alice Lou May 2022 Melanie J. Filiatrault Mary Ann Karp ABSTRACT Dickeya spp. are common plant pathogens associated with bacterial soft rot, potato blackleg, and slow wilt, which are plant diseases that account for major losses in the agricultural industry. The diseases caused by these bacterial species are not yet fully managed with existing techniques, and new approaches need to be considered to minimize future crop loss. Previous research has shown that the inorganic salt potassium tetraborate tetrahydrate (PTB) can inhibit the growth of Dickeya species; however, disk diffusion assays result in a unique phenotype with two zones of inhibition. In this study, I investigated the effects of PTB on the growth of four Dickeya spp. I hypothesized that the production of phage is responsible for the two zones of inhibition. I used disk diffusion assays and growth curves to confirm the impact of PTB on Dickeya and attempted to directly isolate phage from the strains. To determine the mechanism of action of PTB, I used Tn-Seq libraries to determine which genes are required for growth in the presence of PTB. Tn- Seq libraries showed that different Dickeya strains shared seven overlapping genes including stress-related genes that increase bacterial resistance to PTB. I used gene expression studies to determine the changes in gene expression that result from PTB exposure. Preliminary results showed that exposure to PTB induces the expression of stress-related genes in Dickeya to increase survival in the presence of the compound. Further research is needed to better understand the implications of observed changes in bacterial gene expression. INTRODUCTION Members of the Pectobacteriaceae, a family of Gram-negative bacteria, are known to cause bacterial soft rot and potato blackleg among other diseases which are destructive to many crops in the fields [1, 2, 3]. It has been reported that bacterial soft rot losses after crop harvest account for up to a third of total loss, significantly greater than any other disease [4]. The severe losses in agriculture yield from soft-rot plant pathogens make the study of these soft-rot pathogens crucial to the success of the agricultural industry. Soft-rot diseases are particularly detrimental to potatoes, the fourth most important crop in the world, causing up to two-thirds of losses [5]. Soft-rot diseases are currently largely managed by good agricultural and handling practices, but better prevention mechanisms are needed when trying to sustain the food needs of a growing population [6]. Although there are currently several management options used to control the bacteria, these options are limited, and alternative methods need to be considered. Existing management options include cultivation measures and hygienic practices such as using clean seed and planting in pathogen-free areas, optimizing storage conditions to decrease development and spread of disease, and physical seed treatments with heat and UV radiation and chemical treatments primarily with antibiotics [7]. Other potential chemical treatment options to prevent infection are being explored, and it has been reported that inorganic salts inhibit the growth of Pectobacterium, a genus within Pectobacteriaceae, the same family as Dickeya [8]. Not only does the agricultural industry seek effective methods to contain these pests, but there is also understandable concern about the chemicals used to treat crops, specifically regarding their safety for consumption and environmental impact. The inorganic salt potassium tetraborate tetrahydrate (PTB) has been shown to be effective against Pectobacterium in treating soft rot [9]. PTB is used in many other industries beyond agriculture including oil and gas, and has been found to be anti-oxidative, non-genotoxic, and overall environmentally and biologically safe [10]. My lab previously studied the effects of PTB on inhibiting the growth of two Pectobacteriaceae genera: Pectobacterium and Dickeya – PTB was found to inhibit growth of Pectobacterium spp., but the chemical was less effective at inhibiting growth of Dickeya. An interesting double-ring-of-inhibition phenotype was observed with Dickeya in diffusion assays using agar plates [11]. This phenotype has not been previously seen in Dickeya or other bacteria. This study explored many questions pertaining to Dickeya’s unique response to PTB and PTB’s mechanism of action. If PTB is found to be an effective inhibitor of soft rot diseases, it could be considered for further investigation as a potential chemical treatment option for crops in storage and fields. The mechanism of action of PTB is not yet fully known but understanding how the chemical affects bacterial growth is crucial to uncovering its potential for agricultural use. Previous research found that soft-rot Pectobacteriaceae including both Pectobacterium and Dickeya may have phages [12, 13, 14] and phage-like elements [15] in their genome that contribute to bacterial virulence. The presence of prophages can be extremely beneficial to bacterial fitness and can introduce new traits into the host [16, 17]. These new traits can alter the bacterial genome in many ways, one of which is by changing the bacteria’s pathogenicity and responses to stress [18, 19, 20]. Stress conditions can cause phage induction and kill the bacterial host [21]. Although the details are not well understood, phage present in plant-pathogenic bacteria can carry genes that alter plant pathogenicity [22]. The main objective of my research was to investigate the effect of PTB on growth and gene expression in Dickeya spp. I examined possible reasons for the unique disk diffusion phenotype by testing a larger collection of Dickeya strains and exploring several hypotheses tailored to possible reasons for the phenotype displayed by Dickeya when exposed to PTB. My hypotheses are as follows: Hypothesis 1: The inner ring represents bacteria that are resistant to PTB. Hypothesis 2: The zone of clearing is the result of phage-induced gene expression. Hypothesis 3: The genes responsible for sensitivity or resistance in Dickeya are different from those found in Pectobacterium. Hypothesis 4: The presence of PTB induces production of phage by Dickeya spp. MATERIALS AND METHODS Bacterial Strains Dickeya dadantii 3937, Dickeya dianthicola ME23, Dickeya dianthicola 67-19 and Dickeya fangzhongdai 643-a were cultured on lysogeny broth (LB) agar plates or in LB broth (10 g tryptone, 5 g yeast extract, and 10 g NaCl per 1 L) [23] at 28 °C. Antibacterial Activity of PTB Disk diffusion assay was used to test the antimicrobial activity of PTB on the four strains of Dickeya and to confirm their double-ring phenotype [11]. Bacterial suspensions grown overnight were adjusted to 2×108 c.f.u. ml−1 (OD600 = 0.2) and spread on the plates using sterile cotton swabs, and one sterile paper filter disk (6 mm in diameter; GE Healthcare) loaded with 20 µl filtered 500 mM PTB solution was put on each plate. Plates were incubated at 28°C for 24 hours after which pictures were taken. Direct Phage Isolation I used several different protocols in my attempts to isolate phage from the bacteria in the double zone (Fig. 1) and bacteria grown in liquid culture with PTB. These protocols included a phage genomic DNA extraction (modified Promega Wizard method; Promega, Madison, WI, USA) as well as several adaptations involving chloroform [24, 25, 26]. Growth Curve Growth curves were performed using a Synergy 2 multi-mode microplate reader [11]. Overnight cultures of the four strains of Dickeya were grown in LB and adjusted to 2×108 c.f.u. ml−1 (OD600 = 0.2). 200 µl of adjusted overnight culture with PTB concentrations of 0 mM, 1.88 mM, 3.75 mM, 7.5 mM, and 15 mM were measured at approximately 25 °C in for up to 16 h at 30 min intervals. Concentrations were chosen based on growth curves performed by Liu and Filiatrault (2020) on Pectobacterium [11]. Six technical replicates and three biological replicates were performed for each treatment. The vertical lines represent the standard error of the mean. Tn-Seq Libraries I used previously constructed randomly barcoded Tn-Seq libraries to identify genes of interest in three strains of Dickeya: D. dadantii 3937, D. dianthicola ME23, and D. dianthicola 67-19. The libraries were prepared by Tyler Helmann [27]. I recovered the libraries in LB with kanamycin and incubated them at 28 °C for 7 hours until reaching an OD600 of 0.5-0.7. During that time, 24- well plates were prepared with LB and PTB concentrations of 0 mM, 1.88 mM, 3.75 mM, and 7.5 mM. The libraries were inoculated in the 24-well plates and grown overnight. Genomic DNA was extracted using the Monarch Genomic DNA Purification Kit (New England Biolabs, Ipswich, MA, USA). Helmann performed a PCR on the extracted DNA to amplify the barcode sequence and index each sample, and then pooled all the samples to submit to the Biotechnology Resource Center (BRC) Genomics Facility at the Cornell Institute of Biotechnology for Illumina sequencing. The libraries had been previously mapped [27]. Helmann analyzed Tn-Seq mapping data using the FEBA RB-TnSeq analysis pipeline to identify genes for each strain that are important for growth in PTB [27, 28]. Real-Time Quantitative PCR Three strains of Dickeya, D. dadantii 3937, D. dianthicola ME23, and D. dianthicola 67-19, were grown overnight. The next day, half of the bacteria were exposed to 3.75 mM PTB and cultured for another hour while the other half also were cultured for another hour without PTB. The PTB concentration used was selected based on growth curve results. RNA from bacteria grown with and without PTB were used in the qRT-PCR experiments to determine differences in gene expression. RNA was extracted using the Qiagen RNeasy kit (Hilden, Germany), treated with Ambion DNase I (Thermo Fisher, Waltham, MA, USA), and cleaned using the Zymo Clean and Concentrate kit (Irvine, CA, USA). cDNA was made from RNA using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA), and the resulting cDNA was quantified and diluted to 100 ng/μL. I used the SsoAdvanced Universal SYBR Green Supermix instruction manual (Bio-Rad) for real time PCR reactions. Five genes selected from Tn-Seq results were evaluated. For D. dadantii 3937, the genes were: 1) Mannose-1-phosphate guanylyltransferase/mannose-6- phosphate isomerase, 2) phosphomannomutase/phosphoglucomutase, 3) GDP-mannose 4,6- dehydratase, 4) WbeA, and 5) sigma E protease regulator RseP. For D. dianthicola 67-19, the genes were: 1) GDP-mannose 4,6-dehydratase, 2) phosphomannomutase CpsG, 3) mannose-1- phosphate guanylyltransferase/mannose-6-phosphate isomerase, 4) envelope stress response regulator transcription factor CpxR, and 5) microconductance mechanosensitive channel MscM. The reactions were set up with SYBR Green Supermix at 1x, 200 nM of each primer, and 100 ng of cDNA. The thermal cycling protocol was as follows: denaturation at 95 °C for 10 seconds, annealing and extension at 52 °C for 30 seconds, and cycled through 40 cycles on a Bio-Rad Connect. Due to time constraints, qRT-PCR was performed on RNA isolated from two strains: D. dadantii 3937 and D. dianthicola 67-19. Three technical replicates and three biological replicates were performed for each treatment. Data was analyzed using the delta delta Ct analysis [29], and the vertical lines represent the standard deviation of the mean. RESULTS Antibacterial Activity of PTB Disk diffusion assays were used to test the antimicrobial activity of PTB on the four strains of Dickeya to determine whether the phenotype was conserved among species and if colonies growing in the presence of PTB developed resistance. It was found that all strains demonstrated the double-ring phenotype, suggesting that the phenotype is conserved in multiple Dickeya species (Fig. 1). To determine if the bacteria growing between the two zones of clearing are resistant, I isolated the bacteria and repeated the same disk diffusion assay. The disk diffusion assay for bacteria taken from the area between the two zones (bacterial growth between red arrow, inner zone, and black arrow, outer zone) gave phenotypically similar results as bacteria not previously exposed to PTB (Fig. 2). This assay ruled out the possibility PTB had led to the selection of PTB resistant bacteria and rejected hypothesis 1 – had there been evolution, the bacteria between the two zones would all be resistant to PTB in the disk diffusion assay. No colonies were found in the zone of clearing. Since the same phenotype with two zones of inhibition was observed even when using the bacteria grown between the two zones of the original assay, I concluded that the bacteria between the two zones were still sensitive to PTB and that the PTB was not selecting for resistant bacteria. Direct Phage Isolation I attempted to directly isolate phage from all four Dickeya strains to test Hypothesis 2 and determine whether the presence of PTB induces production of phage by Dickeya spp. I followed a phage genomic DNA extraction (modified Promega Wizard method; Promega, Madison, WI, USA) and several adaptations [24, 25, 26], all of which failed to produce any sort of pellet after centrifugation. My phage isolation attempts were unsuccessful, and I was unable to extract phage DNA using any of the protocols. Many variables were in play with direct phage isolation – it was possible that the phage was not released from the bacteria I used, and I instead turned to gene expression experiments (qRT-PCR) and Tn-Seq to investigate phage presence. Growth Curve Growth curves were performed to determine the minimal inhibitory concentration (MIC) of PTB at which each Dickeya strain cannot grow. All strains grew in cultures lacking PTB, and all four Dickeya strains were found to be sensitive to PTB depending on the concentration (Fig. 3). 15 mM PTB showed to completely inhibit the growth of D. dadantii 3937 and D. fangzhongdai 643-a while 7.5 mM PTB was sufficient to inhibit the growth of D. dianthicola ME23 and D. dianthicola 67-19 (Fig. 3). While the double-ring phenotype of interest cannot be observed using growth curves, the MIC was used to guide the concentrations tested in the Tn-Seq experiments. Tn-Seq Libraries Tn-Seq libraries were used to identify genes in Dickeya important for growth in PTB. The genes identified were compared to genes found in PTB-resistant Pectobacterium [11] and were used in qRT-PCR to evaluate changes in gene expression. The libraries revealed several genes that when disrupted improve growth in PTB and several genes that when disrupted are detrimental to growth in PTB (Table 1). All experiments passed quality check, and the genes shown in Table 1 were all statistically significant. Normalized gene fitness (fit) values were used to sort the genes: a positive fit value would indicate the that the strain is more resistant to PTB, and the disruption in the gene improved survival while a negative fit value would indicate that the strain is more sensitive to PTB, and the disruption in the gene was detrimental to the bacterial strain. In D. dadantii 3937, nine genes improved growth in PTB when disrupted, and 14 genes were detrimental to growth in PTB when disrupted; in D. dianthicola ME23, six genes improved growth in PTB when disrupted, and nine genes were detrimental to growth in PTB when disrupted; in D. dianthicola 67-19, five genes improved growth in PTB when disrupted, and five genes were detrimental to growth in PTB when disrupted (Table 1). Seven genes were shared between at least two of the three strains tested including mannose-1 phosphate guanylyltransferase/mannose 6-phosphate isomerase, phosphomannomutase/phosphoglucomutase, GDP-mannose 4,6-dehydratase, microconductance mechanosensitive channel MscM, and envelope stress response regulator transcription factor CpxR (Fig. 4). The genes related to sensitivity and resistance in Dickeya are different from those found in Pectobacterium [11] which confirms Hypothesis 3. Dickeya and Pectobacterium respond to PTB through different mechanisms – the genes found in Dickeya can then be used to study differences in gene expression and potential overlap with phage-related genes. Real-Time Quantitative PCR To track the differences in gene expression between PTB-exposed bacteria and non-exposed bacteria, qRT-PCR was performed using the genes that were identified using Tn-Seq (Table 4). I selected five genes from the Tn-Seq experiments to use as targets for qRT-PCR, and the resulting data was analyzed using the delta delta Ct analysis [29]. Due to time constraints, I was only able to run each experiment once and for only two strains of Dickeya. D. dadantii 3937 and D. dianthicola 67-19 varied greatly in gene expression. Unexpectedly, all five genes tested for D. dianthicola 67-19 had fold changes in gene expression < 1, which would mean that all five genes demonstrated reduced expression in PTB-treated samples (Fig. 5). This result did not agree with my expectations since three of the genes tested are detrimental to growth in PTB when disrupted (Table 1). DISCUSSION Dickeya displayed a unique response to PTB in disk diffusion assays by showing two distinct rings of clearing (Fig. 1). The observed phenotype was hypothesized to potentially be the result of phage production as a response to PTB exposure. Previous research has found Dickeya to contain phages [11, 12, 13] and phage-like elements [14], which could impact bacterial fitness when exposed to a stressor like PTB. All four strains reacted similarly in the disk diffusion assays and were sensitive to PTB in LB, which suggests that response to PTB may be a conserved mechanism. Through multiple disk diffusion assays and the Tn-Seq experiments, I determined that PTB exposure did not select for resistant Dickeya, and the genes responsible for PTB sensitivity or resistance in Dickeya did not overlap with mutated Pectobacterium genes from Liu and Filiatrault (2020) [11]. Further research can be done to more closely investigate these differences. The Tn-Seq experiments provided important information about genes involved in Dickeya resistance and sensitivity to PTB. Tn-Seq experiments showed that Dickeya strains shared several genes of interest, many of which were stress-related genes that may help increase bacterial resistance to PTB. The double-ring phenotype could have been caused by changes in gene expression at different PTB concentrations, but more research is needed to draw conclusions. Stress-related genes are likely required for growth in PTB; borate has been shown to inhibit growth and induce stress responses of certain pathogens [30]. Bacteria respond to stress in a variety of ways, which can include inducing of expression of different genes relating to survival and virulence. Changes in gene expression related to mannose-metabolism have been previously observed in pathogenic bacteria as a response to osmotic stress [31]. The disturbance of mannose-metabolism in response to salinity is consistent with the observed importance of mannose-related genes for growth in PTB (Table 1). The genes disrupted in Dickeya were different than the mutated genes Liu and Filiatrault (2020) found through whole-genome sequencing of PTB-resistant Pectobacterium mutants which affected peptide chain release from the ribosome [11]. I hypothesized that phage was responsible for Dickeya’s response to PTB exposure but attempts to directly isolate phage were unsuccessful. Phage isolation can be heavily timing dependent – it is possible that my isolation attempts did not align with the timing of phage release or that phage was not released from the bacteria. Further research would be necessary to thoroughly explore phage-related responses of Dickeya to PTB exposure. I was unable to reach any firm conclusions regarding phage production due to time constraints. If a suitable phage-sensitive host can be found, plaque assays can be used to identify phage plaques. Otherwise, continued exploration of gene expression would likely be the preferred route. The Tn-Seq experiments revealed genes related to lipopolysaccharide (LPS) production, which has been found to interact with phage [32], reinforcing the likelihood of phage involvement. A potential next step would be to sequence the bacteria grown between the two zones of clearing using both whole genome sequencing and RNA-Seq to examine any mutations or differences in gene expression from the freezer stock. Another possibility would be to directly locate the phage genes in Dickeya and track expression of specific known phage genes such as that of the transducing phage φEC2 [13]. Overall, more work is necessary to examine PTB as a potential chemical treatment for bacterial infection of crops. Understanding Dickeya’s response to PTB would clarify the double-ring phenotype and help evaluate efficacy of PTB treatment and the ideal conditions under which treatment should be applied. Although present findings showed that all four Dickeya remained sensitive to PTB after treatment (Fig. 2), further experiments should investigate the possibility of developing bacterial resistance after continued exposure to PTB to ensure its success as a plant treatment option. FIGURES AND TABLES Fig. 1. Disk diffusion assay of antibacterial activity of PTB on four strains of Dickeya freezer stocks: D. dadantii 3937, D. dianthicola ME23, D. dianthicola 67-19, and D. fangzhongdai 643-a. The black arrows point to the outer zone of clearing and the red arrows to the inner zone. Fig. 2. Disk diffusion assay of antibacterial activity of PTB on four strains of Dickeya re-streaked from the middle zone of growth between the two zones of clearing in Fig. 1. The black arrows point to the outer zone of clearing and the red arrows to the inner zone. Fig. 3. The growth (OD600) of four strains of Dickeya: D. dadantii 3937, D. dianthicola ME23, D. dianthicola 67-19 and D. fangzhongdai 643-a was measured for up to 16 hours. Each treatment had six replicates and each experiment was repeated at least three times. The most recent (4/30/2021) graphs are shown. Error bars indicate standard error of the mean. Dickeya dadantii 3937 Tn-Seq Genes Locus Id Description DDA3937_RS03410 polysaccharide biosynthesis protein DDA3937_RS03420 sugar transferase DDA3937_RS18655 mannose-1 phosphate guanylyltransferase / mannose 6-phosphate isomerase Genes that improved DDA3937_RS18660 phosphomannomutase / phosphoglucomutase growth in PTB when disrupted DDA3937_RS18665 GDP-mannose 4,6-dehydratase DDA3937_RS18670 ABC transporter permease DDA3937_RS18685 WbeA DDA3937_RS18690 Glycosyltransferase DDA3937_RS20290 transcription/translation regulatory transformer protein RfaH Locus Id Description DDA3937_RS00900 cell division protein ZapB DDA3937_RS01600 outer membrane-stress sensor serine endopeptidase DegS DDA3937_RS02810 DedA family protein DDA3937_RS19930 uroporphyrinogen-III c-methyltransferase DDA3937_RS12215 sodium-potassium/proton antiporter ChaA Genes that were DDA3937_RS05150 sigma E protease regulator RseP detrimental to growth in PTB when DDA3937_RS05640 ATP-dependent Clp endopeptidase proteolytic subunit ClpP disrupted DDA3937_RS05645 ATP-dependent protease ATP-binding subunit ClpX DDA3937_RS15765 RNA polymerase sigma factor RpoE DDA3937_RS06350 replication initiation negative regulator SeqA DDA3937_RS11035 redox-regulated ATPase YchF DDA3937_RS11210 septum site-determining protein minD DDA3937_RS18610 RNA chaperone Hfq DDA3937_RS12490 exoribonuclease II Dickeya dianthicola ME23 Tn-Seq Genes Locus Id Description DZA65_RS19720 mannose-1 phosphate guanylyltransferase / mannose 6-phosphate isomerase Genes that improved DZA65_RS19725 phosphomannomutase / phosphoglucomutase growth in PTB when DZA65_RS19730 GDP-mannose 4,6-dehydratase disrupted DZA65_RS19755 glycosyltransferase DZA65_RS03635 sugar transferase DZA65_RS19750 hypothetical protein Locus Id Description DZA65_RS20165 microconductance mechanosensitive channel MscM DZA65_RS20220 glucose-6-phosphate isomerase Genes that were DZA65_RS21610 envelope stress response regulator transcription factor CpxR detrimental to growth DZA65_RS22635 4-amino-4-deoxy-L-arabinose-phosphoundecaprenol flippase subunit ArnE in PTB when DZA65_RS22640 4-amino-4-deoxy-L-arabinose-phospho-UDP flippase disrupted DZA65_RS00800 O-antigen ligase family protein DZA65_RS01525 LPS export ABC transporter periplasmic protein LptC DZA65_RS09455 DUF3413 domain-containing protein DZA65_RS04310 Dyp-type peroxidase Dickeya dianthicola 67-19 Tn-Seq Genes Locus Id Description HGI48_RS18585 mannose-1 phosphate guanylyltransferase / mannose 6-phosphate isomerase Genes that improved HGI48_RS18590 phosphomannomutase CpsG growth in PTB when disrupted HGI48_RS18595 GDP-mannose 4,6-dehydratase HGI48_RS06765 dTDP-4-dehydrorhamnose 3,5-epimerase HGI48_RS03520 sugar transferase Locus Id Description Genes that were HGI48_RS19050 microconductance mechanosensitive channel MscM detrimental to growth HGI48_RS20595 envelope stress response regulator transcription factor CpxR in PTB when HGI48_RS20045 tyrosine recombinase XerC disrupted HGI48_RS05730 SmdA family multidrug ABC transporter permease/ATP-binding protein HGI48_RS01595 DnaA initiator-associating protein DiaA Table 1. Genes found in the three Dickeya strains constructed for Tn-Seq libraries. Fig. 4. Venn diagram of genes identified from Tn-Seq experiments. All experiments passed QC. Genes shown are all genes where |fit| > 2 and |t| > 4 and are sorted by fit value into genes that improved growth in PTB when disrupted (green) and genes that were detrimental to growth in PTB when disrupted (red). Fig. 5. qRT-PCR fold change in gene expression for D. dadantii 3937 and D. dianthicola 67-19. The fold change in gene expression was calculated as a ratio of the gene expression of PTB-treated samples over the gene expression of non-treated samples. A fold change in gene expression < 1 would indicate that PTB-treated samples decreased expression of the gene of interest whereas a fold change in gene expression > 1 would indicate that PTB-treated samples increased expression of the gene of interest. Error bars indicate standard deviation of the mean. ACKNOWLEDGEMENTS First and foremost, I would like to sincerely thank my faculty advisor Melanie Filiatrault and my mentor Mary Ann Karp for their unconditional support throughout my research journey. I would also like to thank Tyler Helmann for his Tn-Seq libraries, Wei Zhang for her assistance with RT- qPCR, Yingyu Liu for welcoming me into the world of research, and everyone in the lab for always being so accommodating and willing to offer a helping hand. REFERENCES 1. Charkowski, A. O. (2018). The changing face of bacterial soft-rot diseases. Annual Review of Phytopathology, 56(1), 269–288. https://doi.org/10.1146/annurev-phyto- 080417-045906 2. Toth, I. K., Barny, M., Brurberg, M. B., Condemine, G., Czajkowski, R., Elphinstone, J. G., Helias, V., Johnson, S. B., Moleleki, L. N., Pirhonen, M., Rossmann, S., Tsror, L., van der Waals, J. E., van der Wolf, J. M., Van Gijsegem, F., & Yedidia, I. (2021a). Pectobacterium and Dickeya: Environment to disease development. In F. Van Gijsegem, J. M. van der Wolf, & I. K. Toth (Eds.), Plant Diseases Caused by Dickeya and Pectobacterium Species (pp. 39–84). Springer International Publishing. https://doi.org/10.1007/978-3-030-61459-1_3 3. Toth, I. K., van der Wolf, J. M., Saddler, G., Lojkowska, E., Hélias, V., Pirhonen, M., Tsror Lahkim, L., & Elphinstone, J. G. (2011). Dickeya species: An emerging problem for potato production in Europe: Dickeya spp. on potato in Europe. Plant Pathology, 60(3), 385–399. https://doi.org/10.1111/j.1365-3059.2011.02427.x 4. Agrios, G. N. (2005). Chapter 12—Plant diseases caused by prokaryotes: Bacteria and mollicutes. In Plant Pathology (5th ed., pp. 615–703). Elsevier Academic Press. http://site.ebrary.com/id/10169629 5. Mantsebo, C. C., Mazarura, U., Goss, M., & Ngadze, E. (2014). The epidemiology of Pectobacterium and Dickeya species and the role of calcium in postharvest soft rot infection of potato (Solanum tuberosum) caused by the pathogens: A review. African Journal of Agricultural Research, 9(19), 1509–1515. https://doi.org/10.5897/AJAR2013.8558 6. Olsen, N., & Nolte, P. (2011, June). Cleaning and Disinfecting Potato Equipment and Storage Facilities. CIS 1180; University of Idaho Extension. https://seedcert.oregonstate.edu/sites/seedcert.oregonstate.edu/files/pdfs/cis1180.pdf 7. van der Wolf, J. M., De Boer, S. H., Czajkowski, R., Cahill, G., Van Gijsegem, F., Davey, T., Dupuis, B., Ellicott, J., Jafra, S., Kooman, M., Toth, I. K., Tsror, L., Yedidia, I., & van der Waals, J. E. (2021). Management of diseases caused by Pectobacterium and Dickeya species. In F. Van Gijsegem, J. M. van der Wolf, & I. K. Toth (Eds.), Plant Diseases Caused by Dickeya and Pectobacterium Species (pp. 175–214). Springer International Publishing. https://doi.org/10.1007/978-3-030-61459-1_6 8. Yaganza, E.-S., Tweddell, R. J., & Arul, J. (2009). Physicochemical Basis for the Inhibitory Effects of Organic and Inorganic Salts on the Growth of Pectobacterium carotovorum subsp. Carotovorum and Pectobacterium atrosepticum. Applied and Environmental Microbiology, 75(5), 1465–1469. https://doi.org/10.1128/AEM.02447-08 9. Ahmed, F. A., Arif, M., & Alvarez, A. M. (2017). Antibacterial Effect of Potassium Tetraborate Tetrahydrate against Soft Rot Disease Agent Pectobacterium carotovorum in Tomato. Frontiers in Microbiology, 8, 1728. https://doi.org/10.3389/fmicb.2017.01728 10. Çelikezen, F. Ç., Turkez, H., Togar, B., & Izgi, M. S. (2014). DNA damaging and biochemical effects of potassium tetraborate. EXCLI Journal ; Vol. 13, 2014. https://doi.org/10.17877/DE290R-15704 11. Liu, Y., & Filiatrault, M. J. (2020). Antibacterial activity and mode of action of potassium tetraborate tetrahydrate against soft-rot bacterial plant pathogens. Microbiology, 166(9), 837–848. https://doi.org/10.1099/mic.0.000948 12. Czajkowski, R. (2016). Bacteriophages of Soft Rot Enterobacteriaceae—A minireview. FEMS Microbiology Letters, 363(2), fnv230. https://doi.org/10.1093/femsle/fnv230 13. Adriaenssens, E. M., Van Vaerenbergh, J., Vandenheuvel, D., Dunon, V., Ceyssens, P.-J., De Proft, M., Kropinski, A. M., Noben, J.-P., Maes, M., & Lavigne, R. (2012). T4-related bacteriophage limestone isolates for the control of soft rot on potato caused by ‘Dickeya solani.’ PLoS ONE, 7(3), e33227. https://doi.org/10.1371/journal.pone.0033227 14. Resibois, A., Colet, M., Faelen, M., Schoonejans, E., & Toussaint, A. (1984). ΦEC2, a new generalized transducing phage of Erwinia chrysanthemi. Virology, 137(1), 102–112. https://doi.org/10.1016/0042-6822(84)90013-8 15. Czajkowski, R. (2019). May the phage be with you? Prophage-like elements in the genomes of soft rot Pectobacteriaceae: Pectobacterium spp. And Dickeya spp. Frontiers in Microbiology, 10, 138. https://doi.org/10.3389/fmicb.2019.00138 16. Bondy-Denomy, J., & Davidson, A. R. (2014). When a virus is not a parasite: The beneficial effects of prophages on bacterial fitness. Journal of Microbiology, 52(3), 235– 242. https://doi.org/10.1007/s12275-014-4083-3 17. Nanda, A. M., Thormann, K., & Frunzke, J. (2015). Impact of spontaneous prophage induction on the fitness of bacterial populations and host-microbe interactions. Journal of Bacteriology, 197(3), 410–419. https://doi.org/10.1128/JB.02230-14 18. Hacker, J., & Carniel, E. (2001). Ecological fitness, genomic islands and bacterial pathogenicity: A Darwinian view of the evolution of microbes. EMBO Reports, 2(5), 376–381. https://doi.org/10.1093/embo-reports/kve097 19. Fortier, L.-C., & Sekulovic, O. (2013). Importance of prophages to evolution and virulence of bacterial pathogens. Virulence, 4(5), 354–365. https://doi.org/10.4161/viru.24498 20. Casjens, S. (2003). Prophages and bacterial genomics: What have we learned so far?: Prophage genomics. Molecular Microbiology, 49(2), 277–300. https://doi.org/10.1046/j.1365-2958.2003.03580.x 21. Du Toit, A. (2019). Phage induction in different contexts. Nature Reviews Microbiology, 17(3), 126–127. https://doi.org/10.1038/s41579-019-0150-4 22. Varani, A. M., Monteiro-Vitorello, C. B., Nakaya, H. I., & Van Sluys, M.-A. (2013). The role of prophage in plant-pathogenic bacteria. Annual Review of Phytopathology, 51(1), 429–451. https://doi.org/10.1146/annurev-phyto-081211-173010 23. Bertani, G. (1951). Studies on lysogenesis i: The mode of phage liberation by lysogenic Escherichia coli. Journal of Bacteriology, 62(3), 293–300. https://doi.org/10.1128/jb.62.3.293-300.1951 24. Phage genomic DNA extraction – modified Promega Wizard method. Cpt.tamu.edu. (2011). Retrieved 17 March 2022, from https://cpt.tamu.edu/wordpress/wp- content/uploads/2011/12/Phage-DNA-extraction-modified-Wizard-method-07-12- 20111.pdf. 25. Protocol for Phage DNA Extraction with Phenol:Chloroform. Cpt.tamu.edu. (2018). Retrieved 17 March 2022, from https://cpt.tamu.edu/wordpress/wp- content/uploads/2018/09/Phage-DNA-Extraction-by-PhenolChloroform-Protocol.pdf. 26. Adams, M. H. (1959). Concentration and Purification of Phage. In Bacteriophages (pp. 457–360). essay, Interscience Publishers. Retrieved from https://archive.org/details/bacteriophages00adam/mode/2up. 27. Helmann, T. C., Filiatrault, M. J., & Stodghill, P. V. (2022). Genome-wide identification of genes important for growth of Dickeya dadantii and Dickeya dianthicola in potato (Solanum tuberosum) tubers. Frontiers in Microbiology, 13. https://www.frontiersin.org/article/10.3389/fmicb.2022.778927 28. Wetmore, K. M., Price, M. N., Waters, R. J., Lamson, J. S., He, J., Hoover, C. A., Blow, M. J., Bristow, J., Butland, G., Arkin, A. P., & Deutschbauer, A. (2015). Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons. MBio, 6(3), e00306-15. https://doi.org/10.1128/mBio.00306-15 29. Bradburn, S. (2020, December 16). How to perform the delta-delta CT method. Top Tip Bio. Retrieved March 17, 2022, from https://toptipbio.com/delta-delta-ct-pcr/ 30. He, S. T., Chen, T. T., Xu, X. B., Zhang, Z. K., Song, H. C., Song, H. M., Meng, L. H., Zhou, P., & Shi, X. Q. (2019). Proteomic analysis of the mango anthracnose pathogen Colletotrichum gloeosporioides treated with borate highlights distinct mitochondrial response mechanisms. Plant Pathology, 68(7), 1369–1380. https://doi.org/10.1111/ppa.13058 31. Chowdhury, R., Sahu, G. K., & Das, J. (1996). Stress response in pathogenic bacteria. Journal of Biosciences, 21(2), 149–160. https://doi.org/10.1007/BF02703105 32. Andres, D., Hanke, C., Baxa, U., Seul, A., Barbirz, S., & Seckler, R. (2010). Tailspike interactions with lipopolysaccharide effect dna ejection from phage p22 particles in vitro. Journal of Biological Chemistry, 285(47), 36768–36775. https://doi.org/10.1074/jbc.M110.169003