Evaluating the contribution of pyoverdine to the anti-Phytophthora activity of two potato-associated Pseudomonas strains

Evaluating the contribution of pyoverdine to the anti-Phytophthora activity of two potato-associated Pseudomonas strains | 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 Article Evaluating the contribution of pyoverdine to the anti-Phytophthora activity of two potato-associated Pseudomonas strains Livia Jerjen, Floriane L’Haridon, Laure Weisskopf This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7139568/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The oomycete Phytophthora infestans has been causing detrimental yield losses over the last 200 years and to this day, controlling measures heavily rely on synthetic pesticides. In view of their environmental toxicity, biological control represents an attractive alternative to fight this pathogen. Pseudomonas strains are known to produce a large arsenal of secondary metabolites conferring protection against several crop diseases. Next to biocontrol traits causing direct pathogen inhibition, such as antibiotics and toxins, siderophores are considered important mediators of competitive inhibition of plant pathogens by Pseudomonas . However, whether siderophore production plays any role in the biocontrol of the late blight causing agent P. infestans has not yet been investigated. In this study, we focused on two Pseudomonas strains, R32 and R47, which have been previously characterized as successful antagonists against P. infestans . Both strains produce pyoverdine, thus the aim of this study was to evaluate the role of pyoverdine in the inhibition of P. infestans in both strains. For this purpose, we created pyoverdine-deficient mutants by knocking-out pvdE , the periplasmic ferribactin exporter. We did this in both wild-type and HCN-deletion backgrounds for the two strains. These mutants were then tested for loss of antagonistic activity against P. infestans in several in vitro assays targeting different developmental stages of the pathogen life cycle, as well as in leaf disc assays to assess the relevance of pyoverdine in planta. Our results indicate that pyoverdine plays a different role in both Pseudomonas strains: in leaf disc assays, lack of pyoverdine completely suppressed the ability of R47 to restrict symptom development, but it increased the protective efficacy of R32. In this latter strain, the lack of pyoverdine alone did not diminish its ability to inhibit the pathogen‘s mycelium or spores, but when combined to the loss of HCN, it either led to a complete loss of inhibition in spore assays, or to stronger inhibition in mycelium assays. These results suggest an interplay between HCN and pyoverdine and the upregulation of a yet unknown mechanism underlying the higher in planta protective efficacy observed in R32 pyoverdine-deficient mutants. Biological sciences/Microbiology Biological sciences/Plant sciences Pseudomonas Phytophthora infestans hydrogen cyanide pyoverdine siderophores biocontrol potato Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Crops are exposed to a variety of pathogens that can cause severe yield losses. In potato in particular, Phytophthora infestans stands out as a challenging pathogen worldwide (Kamoun et al., 2015). This oomycete, causing late blight in Solanaceae crops, can destroy a potato field in 10–15 days only (Rakotonindraina et al., 2012). It is considered the most threatening potato pathogen due to its rapid evolution, high adaptation capacity and fast asexual reproduction cycle involving two types of spores; sporangia that germinate directly under warmer conditions, and motile zoospores that are released from sporangia when temperatures are colder (Fry et al., 2015). Current crop protection practices heavily rely on copper-based and synthetic pesticides, the latter being not only toxic but also prone to resistance development in such a rapidly evolving pathogen as P. infestans (Leesutthiphonchai et al., 2018). In contrast, biocontrol agents are less likely to trigger resistance development in pathogens due to their multi-target modes of action (Ivanov et al., 2021). In bacteria, these modes of action can encompass induction of plant resistance, or more direct mechanisms like antibiosis and competition for resources and space (Wang & Long, 2023). The latter includes also the competition for iron, which despite its abundance in the environment, is difficult to acquire in soils due to its insolubility in the ferric state (Owen & Ackerley, 2011). Microorganisms are able to produce iron-scavenging molecules, called siderophores, that allow them to take up the insoluble ion from the soil and make them great competitors for iron (Schalk, 2008), a feature that has been shown to play an important role in biocontrol of plant pathogens (Liu et al., 2021; Ran et al., 2005). Previous research in our group has focused on two Pseudomonas strains showing promise for the biological control of late blight: Pseudomonas sp. R32, which belongs to the P. putida group, and Pseudomonas sp. R47, which belongs to the P. protegens group. Both strains showed strong inhibitory activity on different developmental stages of P. infestans (mycelium, sporangia, zoospores) (Anand et al. 2020, 2023; Hunziker et al. 2015). A comparative genomics approach identified putative biocontrol traits and mechanisms in these two strains (De Vrieze et al., 2018, 2020; Hunziker et al., 2015), among which the production of phenazines by Pseudomonas sp. R47, and the production of hydrogen cyanide (HCN) and pyoverdine by both strains. The role of hydrogen cyanide in both strains’ anti- Phytophthora activity has been investigated previously (Anand et al., 2020), and was shown to affect mycelial growth but not spore germination, nor disease progression in leaf discs. In contrast, the importance of pyoverdine for the anti- Phytophthora activity has not been evaluated yet. Pyoverdine was first described as a siderophore in 1978 (Meyer & Hornsperger, 1978). Since then, its role in iron competition has been well established (Schalk & Guillon, 2013; Visca et al., 2007) and its synthesis and regulation are also well-studied (Henríquez et al., 2019; Ringel & Brüser, 2018; Yeterian et al., 2010). All pyoverdines are composed of a fluorescent dihydroquinoline-type chromophore which is linked to a strain-specific peptide chain and a side chain at the C3 position (Schalk & Guillon, 2013). Pyoverdine’s complex synthesis is regulated at multiple levels; by the global two-component system regulator GacS/GacA, directly by intracellular iron concentrations through the ferric uptake regulator (FUR) (Ochsner et al., 1995), by several transcriptional LysR-type regulators (Kang & Kirienko, 2017) and at post-transcriptional level by FpvR (Edgar et al., 2014). The synthesis involves a total of twelve non-ribosomal peptide synthetases (NRPS) and other proteins (Visca et al., 2007). Although siderophores in general, and pyoverdine in particular, have been shown to play a role in the biological control of plant diseases (Liu et al., 2021; Ran et al., 2005), the extent of pyoverdine’s contribution to the anti- Phytophthora properties of Pseudomonas strains remains unknown. To fill this gap of knowledge, we created pyoverdine mutants in both wild-types and HCN-deletion mutants of the two Pseudomonas biocontrol strains R32 and R47. We selected PvdE , which encodes an ATP-binding cassette (ABC) transporter that exports pyoverdine’s precursor ferribactin from the cytosol into the periplasm for the maturation process, as a target for mutagenesis, since knocking-out this gene has been reported to abolish pyoverdine production in P. aeruginosa (Yeterian et al., 2010). We characterized the ability of these mutants to inhibit different developmental stages of P. infestans in in vitro experiments, as well as their impact on disease progression in planta , to gain a detailed understanding of pyoverdine’s putative role in the inhibition of the causative agent of late blight by two different potato-associated Pseudomonas strains. Material and Methods Strains and culture conditions Pseudomonas sp. R32 and Pseudomonas sp. R47 were originally isolated from the rhizosphere of potato plants (Hunziker et al., 2015). The two wild-types and their respective mutants were routinely cultured on LB-Agar plates with 10 µM rifampicin at 28°C. LB agar plates were prepared with 12.5 g.L − 1 LB Broth Miller (Roth), 10 g.L − 1 LB Broth Lennox (Roth) and 15 g.L − 1 of Agar-Agar-Kobe I (Roth), which were dissolved in distilled water and autoclaved. Liquid cultures were incubated overnight at 180 rpm at 28°C. KB medium was prepared by mixing 20 g.L − 1 proteose peptone #3 (Gibco), 1.5 g.L − 1 K 2 HPO 4 (Roth), 1.5g.L − 1 MgSO 4 x 7H 2 0 (Roth), 10 ml.L − 1 Glycerol (Reactolab) and 15 g.L − 1 Agar-agar Kobe I (Roth) for solid medium, dissolving them in distilled water and autoclaving. P. infestans strains Rec01, 44 and 208m2 (GFP-tagged strain, (Si-Ammour, Mauch-Mani, and Mauch 2003)) were routinely grown at 18°C on V8-Agar plates. This medium was prepared by diluting V8 100 % hot spicy vegetable juice at 100 ml.L 1 in distilled water, and adding of 1 g.L − 1 of CaCO 3 (Roth) and 15 g.L − 1 of agar. Liquid V8 medium was filtered in some experiments with 0.22 µm filters (Millex) after autoclaving in order to get rid of debris. After a maximum of seven passages on plates, P. infestans was passaged on potato tubers to keep its virulence. Generation of knock-out (KO) mutants in Pseudomonas R32 and R47 We followed the same protocol as described earlier (Anand et al., 2020) to generate pvdE KO mutants in both R32 and R47 wild-type and ∆ hcn strains. Since for R32, the attempts to amplify the gene fragments for the first cloning step were unsuccessful, these sequences were produced synthetically by Eurofins (© Eurofins Scientific 2023). Please refer to Table S1 for the list of plasmids and strains, and Table S2 for the primers used and generated. Growth curves and monitoring of pyoverdine production over time Overnight bacterial cultures in LB were centrifuged at 5000 rpm, the supernatant removed, and the pellet resuspended in 0.9% NaCl (Roth). After a second centrifugation step, the washing supernatant was removed and resuspended in the same solution. OD 600 was adjusted to 1 and in a 96-well plate, we mixed 5 µL of the bacteria (or 0.9% NaCl for the negative control) with 195 µL of either liquid LB, KB or filtered V8. We performed triplicates for each sample. Then the plate was incubated during 60 h in the Cytation 5 with shaking at 28°C. Every hour, absorbance at 600 nm and pyoverdine fluorescence (excitation at 405 nm, emission at 460 nm) were measured. Siderophore detection with Chrome azurol S (CAS) CAS media were prepared by mixing 10 mL of iron solution (270 mg.L − 1 FeCl 3 x 6 H2O (Merck) and 1% HCl 1M (Fisher Chemicals) in distilled water) with 50 mL of solution 1 (1.21 g.L − 1 chrome azurol S (Kodak) in distilled water). 40 mL of solution 2 (1,8225 g.L − 1 HDTMA (Sigma) dissolved in distilled water) were added to the first mix, and the final CAS solution was autoclaved. KB-Agar was prepared as described above and before autoclaving, 30.24 g.L − 1 of PIPES PUFFERAN (Roth) were added, the pH was adjusted to 6.8 with NaOH and then 15 g.L − 1 of agar were added. KB-medium was autoclaved, then mixed with the CAS solution in a ratio of 9:1. Overnight bacterial cultures in LB were centrifuged at 5000 rpm, the supernatant removed, and the pellet resuspended in 0.9% NaCl (Roth). After a second centrifugation step, the washing supernatant was removed and resuspended in the same solution. OD 600 was adjusted to 1 and 10 µL of the bacterial suspensions were pipetted onto CAS-KB plates before incubating for 48 h at 28 ° C. Pyoverdine measurements Overnight cultures of bacteria in LB were rinsed in 0.9% NaCl (Roth) as described above and adjusted to OD 600 = 1 in the same solution. In triplicates, we added 5 µL of bacterial suspension (or 0.9% NaCl for the negative control) to 195 µL KB or KB supplemented with FeCl 3 solution (see CAS iron solution above) in 96-well plates (Costar). Parafilmed plates were incubated for 48 h at 28°C under shaking at 120 rpm. Pyoverdine fluorescence (excitation at 405 nm, emission at 460 nm) and OD 600 were measured in a Cytation 5 cell imaging reader (Biotek). Both the negative controls of fluorescence and OD values were subtracted from the measured sample values, and those subsequently normalized to OD 600 = 1. After normalization, mean values and SD were calculated for each sample and statistical analysis was performed using a one-way ANOVA, followed by a Tukey’s test, where ∗=p < 0.05, ∗∗=p < 0.01, and ∗∗∗=p < 0.001. For the assay with FeCl 3 supplementation, we performed a mixed-effect model analysis with Geisser-Greenhouse correction, followed by a Tukey’s test. HCN detection 50 g.L − 1 of copper(II)-ethyl acetoacetate (Aldrich) and 50 g.L − 1 4,4-methylenbis(N,N-dimethylaniline) (Sigma-Aldrich) were dissolved in chloroform (Fisher Chemicals). This solution was then pipetted onto pieces of filter paper (Whatman) that had been cut beforehand. The paper was dried overnight under a fume hood and then stored in a glass container wrapped in aluminum foil at 4°C. Split Petri dishes (Sarstedt) were filled on one side with LB-Agar and left empty on the other side. 10 µL of bacteria at OD 600 = 1 were pipetted onto the LB-Agar and a detection paper placed into the empty dish side. Plates were sealed with parafilm and incubated at 30°C, pictures were taken at 24 h and 48 h after incubation. In vitro dual assays Experiments were performed on plates filled with V8-Agar, which in some experiments was supplemented with FeCl 3 (see CAS iron solution above). Overnight cultures of bacteria in LB were rinsed in 0.9% NaCl (Roth) as described above and adjusted to OD 600 = 1 in the same solution. Three drops of 10 µL of bacterial suspension (or 0.9% NaCl for negative control) were carefully applied symmetrically onto an V8-Agar plate near the border. A plug of P. infestans Rec01 culture was then placed in the middle of the plate. Sealed plates were incubated at 21°C for 12–14 days. Four replicate plates per bacterial strain and six replicate plates for the control ( P. infestans growing alone) were performed per experiment. For the analysis, the area of growth of the pathogen’s mycelium and the area of the bacterial colony were measured with ImageJ (Schneider et al., 2012). The percentage of growth inhibition caused by the bacteria was calculated with the following formula: 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 %=100∗𝐴𝑖/(𝐴𝐶−𝐴𝑏), with inhibition area 𝐴𝑖 = 𝐴𝐶−𝐴𝑏−𝐴𝑡, where 𝐴𝐶= pathogen area in control plates, 𝐴𝑏= bacterial area, 𝐴𝑡= pathogen area. Note that the inhibition area was calculated considering that the bacterial area is not available anymore for the pathogen because of space competition (𝐴𝐶−𝐴𝑏). We performed a mixed-effect model analysis with Geisser-Greenhouse correction, followed by a Tukey’s test, where ∗=p < 0.05, ∗∗=p < 0.01, and ∗∗∗=p < 0.001. Spore assays We performed zoospore release, zoospore germination and sporangia germination assays, and for all three types, an overnight liquid culture of bacteria in LB was rinsed in 0.9% NaCl (Roth) as described above and adjusted to OD 600 = 1 in filtered V8. 50 µL of bacterial suspension were transferred into flat-bottom 96-well plates (Costar). The procedure for each assay differed from here, as detailed below. For the zoospore germination assay, 50 µL of 0.9% NaCl were added to the bacterial suspension. Then, 10 mL ice cold, sterile water were poured onto 2 to 3 week-old P. infestans cultures on V8-agar plates which were placed at 4°C in the dark for 2 h. After this cold shock, the plates were placed at 18°C for 30 min. Zoospores were then collected by aspiring the water from the mycelium surface with a pipette and their concentration adapted to 3x10 5 zoospores.mL − 1 . 40 µL of zoospore solution were added into the wells. The sealed plates were then incubated in the dark at 18°C for 3–4 h. For the zoospore release assay, sporangia were collected by adding 2–3 mL of sterile water onto a 2 to 3 week-old P. infestans plate and scratching off the mycelium from the agar with a glass slide. The mycelium was washed through a filter with sterile water into a 15 mL falcon tube (Cellstar), so that sporangia were separated from the mycelium. The sporangia solution was then adapted to 3x10 5 sporangia.mL − 1 and 40 µL added into wells. 50 µL of ice cold sterile water were added into the wells and the plate was placed at 4°C in the dark for 2 h, then for 30 min at 18°C, still in the dark. For the sporangia germination assays, 50 µL of 0.9% NaCl were added to the wells. Sporangia were collected as described above and 40 µL of the final solution of 3x10 5 sporangia.mL − 1 was added before incubating in humid, dark conditions for 24 h. After the respective incubation times, pictures were taken with the Cytation 5 (Biotek), and then percentages of germination for sporangia and zoospores were calculated by counting germinated vs. non-germinated spores (total spores per replicate ca. 200 spores), and release percentage was calculated by counting full vs. empty sporangia, counting the same number of spores as above per picture. All experiments were performed in technical triplicates. For statistical analysis, we pooled together results from 4 experiments and used a Kruskal-Wallis test followed by a Dunn’s multiple comparisons test performed either on GraphPad Prism or R Studio. For sporangia germination, we measured additionally the length of germ tubes with ImageJ in 2 experiments with each three technical replicates and performed an ANOVA with Tukey’s post-hoc test for statistical analysis. Leaf disc assays Potato plants from the Bintje cultivar were cultivated in a greenhouse for 6–7 weeks. Leaf discs were cut and placed abaxial side up onto a 0.8% water agar plate (5 discs per plate) the day before inoculation and infection. We analyzed 4 plates per bacterial treatment and negative control (20 leaf discs). One leaf disc per plant was used as a non-infected control. Bacteria were cultured overnight in LB, washed in 0.9% NaCl and resuspended at OD 600 = 2 in the same solution. Zoospores were harvested from 2 week-old P. infestans cultures as described above. The zoospore solution was adjusted to 6-8x10 5 spores.mL − 1 . The bacterial suspension and zoospore solution were mixed in a 1:1 ratio and 10 µL of this mixture was applied onto each leaf disc. Plates were then placed into a transparent, closed box with high humidity at 21°C with a light/dark cycle for max. 6 days. Pictures were taken at 4 dpi and 5 dpi. Lesion development was scored at 4 dpi from 0 (no lesion) to 5 (full necrosis) and mycelium development was scored at 5 dpi from 0 (no mycelium) to 5 (fully covered by mycelium). We used a Kruskal-Wallis test followed by a Dunn’s multiple comparisons test for both scores separately. Results Deletion of pvdE leads to decreased secretion of pyoverdine in both R32 and R47 To evaluate the impact of siderophore production on the anti- Phytophthora activity of Pseudomonas R32 and R47, pyoverdine mutants were generated by deleting the pvdE gene in both wild-type and HCN-deletion mutant backgrounds (Anand et al., 2020). This mutation did not impair the strains’ growth (Fig. S1 ). As expected, pyoverdine levels as revealed by fluorescence emission (Kang & Kirienko, 2017) were strongly reduced in single and double mutants of both strains (Fig. 1 A, Fig. S2), although production was not completely abolished in R32. Higher pyoverdine emission was observed in R32 than in R47 and in single ∆ hcn mutants than in the respective wild-type strains, as previously reported (Anand et al., 2023). This was visible in the pyoverdine measurement in liquid cultures of R32 (Fig. 1 A) and in the UV fluorescence emitted on solid culture plates for R47 (Fig. 1 B). To gain a broader view of siderophore production beyond the measurement and visualization of fluorescent molecules, we grew the eight genotypes on CAS plates, on which siderophore-mediated iron depletion can be visualized by an orange halo. In contrast to our expectations, we observed no clear correlation between pyoverdine fluorescence measurements and the size of the orange halo, which suggests that this halo is mediated by the secretion of other siderophores. As expected, pyoverdine production correlated with iron deficiency, although differences were observed between the two strains: R32 gradually decreased its pyoverdine production with increasing iron concentrations but still produced detectable amounts at the highest iron supply tested (27 mg.L − 1 ), while R47 stopped producing pyoverdine already when supplied with 1 mg.L − 1 iron in its growth medium (Fig. 2 A and B). Iron supply decreased the ability to restrict P. infestans mycelial growth in R32, but not in R47 In V8 medium without iron supplementation, we observed no significant loss of activity in single ∆ pvdE mutants compared to the wild-type strains (Fig. 3 A and B, Table S4). While all R47 mutants kept the full inhibition potential of the wild-type, slight modulations were observed in R32, with a small but significant decrease of activity in the single ∆ hcn mutant and a partial rescue of the wild-type phenotype in the double mutant (Fig. 3 A, Table S4). Adding increasing concentrations of iron did not impair R47’s ability to inhibit mycelial growth, suggesting that competition for iron is not involved in this process. In contrast, both wild-type and mutant strains of R32 lost part of their activity with increasing iron supply, suggesting that part of the observed inhibition potential was due to iron deprivation of P. infestans . This was most visible in the absence of HCN. However, ∆ pvdE mutants did not show a more severe activity loss than their corresponding controls (wild-type vs. single ∆ hcn mutant) as could have been expected if pyoverdine had played a role in iron acquisition. On the contrary, the double knock-out seemed to regain part of the wild-type’s inhibitory potential independently of iron supplementation, suggesting the putative upregulation of a yet unknown factor leading to mycelial restriction when both HCN and pyoverdine are no longer produced. It was striking to observe that R32 and its respective mutants were accumulating and secreting a reddish molecule when growing on plates supplemented with high iron concentrations, a phenomenon that was the most intense in the single HCN mutant in R32. In R47, no secretion of this molecule could be observed, only a slightly darker coloration in the HCN mutant and double mutant at the highest iron concentrations. (Fig. 3 B). A phenotype visible in both strains was the decreasing colony diameter with increasing iron concentrations which could be due to a higher agar density mediated by iron addition, and was not linked to toxicity, as cell numbers revealed by CFU counting were not reduced even at the highest iron dose (Fig. S3). Mutants deprived of both HCN and pyoverdine lost their spore inhibition potential in R32, but not in R47. In addition to mycelial growth inhibition, we analyzed whether pyoverdine would play a role in the inhibition of zoospore release (Fig. 4 ), zoospore germination (Fig. 5 ) and sporangia germination (Fig. 6 ). As spore physiology shows higher variability between pathogen strains than mycelial growth, we analyzed three different P. infestans genotypes. Similar results were obtained for the three strains (see Fig. S4, S5 and S6), hence we only present the results obtained with the GFP-labeled strain in Fig. 4 – 6 . In general, R47 showed very little spore inhibition potential compared to R32, which led to strong inhibition even when applied at low cell densities. In both zoospore release and germination, no significant change of activity was observed in the mutant genotypes of R47 compared to the wild-type at both tested concentrations (Fig. 4 A, 4 B, 5 A and 5 B). Although not significant, we could still observe a tendency towards increased antagonistic activity for the R47 HCN single mutant and the double mutant in the zoospore germination (Fig. 5 A and B). In R32, both single mutants kept their full potential and a strong loss of inhibition was observed only for the double mutant. For zoospore release, the effect that was only a tendency at lower bacterial concentration (Fig. 4 A) became a significant loss of inhibition at the higher concentration (Fig. 4 B), where it almost reached the same level of release as in the water control. Regarding the zoospore germination, at both tested concentrations, the difference between the double mutant and all other R32 genotypes was not statistically significant due to high variability between replicates, but a clear trend was visible (Fig. 5 A and B). Since no loss of inhibition was detected for either of the single mutants, this result suggests an interplay or a redundant effect of both biocontrol traits. Sporangia germination yielded a contrasting picture, in which no difference between wild-type and mutants was observed neither in R32 nor in R47 for germination percentages (Fig. 6 A, B, D and E). When looking at germ tube length, neither pyoverdine nor HCN loss affected the inhibition ability of R32 (Fig. 6 C), while for R47, both single and double ∆ hcn mutants showed higher activity than the wild-type, suggesting the upregulation of other active compounds in absence of HCN (Fig. 6 F). Dual loss of HCN and pyoverdine decreased in planta activity in R47 but increased activity in R32 Since infection of the leaf discs was performed with zoospores in direct contact with the bacteria, we expected to have similar results as in the zoospore germination assays. Surprisingly, the results showed opposite trends compared to the in vitro experiment, underlining the differing conditions of in vitro and in planta assays (Fig. 7 ). In R32, the pvdE single mutant displayed a significant increase of protection against P. infestans compared to the wild-type, both when looking at lesion size (Fig. 7 A) or mycelium development (Fig. 7 B). The double mutant also was significantly more protective compared to the wild-type, but not when compared to ∆ hcn , since this mutant already showed a trend towards higher protection. These findings suggest an upregulation of one or several other biocontrol traits in absence of HCN and pyoverdine. In the case of R47, we observed a complete loss of protective activity in all mutant strains compared to the wild-type. In this case, HCN and pyoverdine appeared to be important traits to protect leaf discs against P. infestans . Discussion To assess the importance of pyoverdine in the biocontrol activity of two Pseudomonas strains against P. infestans , we knocked out pvdE in both wild-type and HCN-deletion mutants. Although Yeterian et al. (2010) reported to prevent pyoverdine production and accumulation in P. aeruginosa by knocking out this gene, we could only achieve the same in one of our two strains. In R32, both the single pvdE mutant and the double mutant still displayed a decreased but measurable pyoverdine level, corresponding approximately to the wild-type level in R47 (Fig. 1 A). While it was still significantly reduced compared to the wild-type in R32, the upcoming question is why we could not achieve a complete depletion of pyoverdine in this strain. Although we cannot exclude a spontaneous diffusion through the membrane, we looked for homologues in the genome of R32 which could take over the transport of ferribactin into the periplasm, but without conclusive results. Although the levels measured in the mutant of R32 might still be biologically relevant, we decided to go further with this mutant, with the hypothesis that a 7-fold reduction in pyoverdine levels (Fig. 1 A) would be enough to assess the relevance of pyoverdine in the strain’s anti- Phytophthora activity. When measuring pyoverdine-specific fluorescence levels, we could clearly observe higher amounts of pyoverdine in R32 than in R47 (Fig. 1 A and 2 A). In contrast, in the fluorescence pictures we can see a more pronounced intensity in R47 (Fig. 1 B and 2 B). Under broad spectrum UV other fluorescent molecules can be visible, which explains the observed discrepancy. The more intense fluorescence for R47 ∆hcn compared to R47 WT could correspond to another fluorescent siderophore, which would be activated by pyoverdine accumulation as a signal and would be repressed by HCN. The reduced levels of pyoverdine levels in all four pvdE mutants were not reflected in a reduced halo size in the CAS assay (Fig. 1 B), which is a clear indicator that both R32 and R47 might produce and secrete one or multiple, yet unknown siderophores, in addition to pyoverdine. The impact of the lack of pyoverdine on the ability to inhibit P. infestans strongly differed between the two Pseudomonas strains In the case of R47, the impact of pyoverdine in mycelium growth inhibition was difficult to assess, since the high inhibitory activity was maintained for all mutants, across all iron supplementation levels (Fig. 3 ). This was likely due to the phenazines produced by R47, which are known to be efficient biocontrol-associated molecules (Biessy & Filion, 2018) and have been previously demonstrated to restrict the mycelial growth of P. infestans (Morrison et al., 2017). Therefore, a putative impact of pyoverdine might have been masked by the presence of phenazines in this strain. Evaluating pyoverdine’s contribution to the inhibition of spore-related developmental stages of P. infestans by R47 was just as difficult, this time for the opposite reason – a very weak or even absent activity in the wild-type despite using higher cell densities than for R32 in these experiments (Figs. 4 – 6 ). This lack of activity differs from earlier experiments (Anand et al., 2020), but was stable across eight biological replicates and all three tested pathogen genotypes. Nevertheless, although we could not observe any significant decrease in germination percentage in R47-exposed sporangia, the length of the germ tube was significantly decreased in contact with R47 wild-type. Loss of pyoverdine did not affect this activity, in contrast to the loss of HCN, which increased it in both single and double mutants (Fig. 6 F). In contrast, the results obtained in planta for R47 and its mutants clearly showed an impact of both pyoverdine and HCN on disease control: while the wild-type protected the leaf discs successfully from lesion and mycelium formation, all three mutants lost their protecting activity (Fig. 7 ), suggesting that both traits are important determinants of in planta protection. Rather than only partially losing their protection, the single mutants were not more efficient than the double mutant, suggesting that in R47, pyoverdine and HCN could regulate each other and/or belong to a common pathway important for biocontrol activity against P. infestans on leaf discs. This suggests that phenazines may not be as important to inhibit mycelium development in planta as in vitro , a tendency that was already observed previously (Morrison et al., 2017). Most importantly, we observed in this experiment a loss of biocontrol activity in the pyoverdine mutants, highlighting the relevance of this siderophore for the control of potato late blight. The loss of pyoverdine could have a direct impact on the iron competition, or a more indirect impact which could be linked to lower iron concentration inside the bacterial cells, which would in turn result in the dysregulation of many regulatory processes involving iron (Crosa, 1997). For R32, the results obtained present themselves quite differently, which underlines that the two strains, although both Pseudomonas displaying antagonistic activity against P. infestans , likely employ different modes of action. In the mycelium growth inhibition under low iron conditions (no added FeCl 3 ), the strongly reduced amount of pyoverdine had no impact on the level of inhibition in both the single and double mutants (Fig. 3 ). This suggests that although iron competition plays a role in the interaction, which was evident from the decreased inhibition observed upon increasing iron supplementation, pyoverdine itself is not relevant for mycelial growth inhibition. More so, if pyoverdine was important for inhibiting this stage of the pathogen, we would expect a cumulative effect of the loss HCN (which resulted in reduced activity in the ∆ hcn mutant) and pyoverdine, but we rather measured a significantly increased activity in the double mutant compared to the ∆ hcn mutant, independently of iron supplementation. This increase in inhibition could be explained by the upregulation of one or multiple other biocontrol traits, such as another siderophore, as suggested above. When looking at the plates supplemented with iron (Fig. 2 B and 3 B), we saw that R32 and its mutants secreted a reddish molecule and produced smaller and thicker colonies. The latter phenomenon of changed morphology was also observed in P. aeruginosa and B. cenocepacia (Berlutti et al., 2005), where increased biofilm formation was reported after iron addition. In zoospore release and zoospore germination, the strong decrease of pyoverdine did not reduce the activity in R32, and neither did the loss of HCN (Figs. 4 and 5 ). However, the loss of both biocontrol traits did result in strong reduction or even complete loss of inhibition activity, suggesting a redundant effect of both traits. We expected results from in planta assays to reflect those obtained in in vitro zoospore germination assays, but it was surprisingly not the case: both single and double mutants of pvdE displayed conferred higher protection than R32 wild-type (Fig. 7 ), suggesting again that in this strain, pyoverdine is not involved in the inhibition of P. infestans , but that its absence would trigger the upregulation of another biocontrol trait, which remains to be identified. Beyond iron competition – a putative regulatory role of pyoverdine? The importance of pyoverdine as a mediator of competition for iron between Pseudomonas and P. infestans was minor in most of our findings, except for the protective ability of R47 in leaf disc assays. This was surprising to us, since pyoverdine is known to have an especially high affinity for iron and is thought to be the main mechanism of iron acquisition in fluorescent Pseudomonas (Poole & Mckay, 2003). Since other studies have reported that pyoverdine was important for biocontrol activity (Liu et al., 2021; Ran et al., 2005; Sass et al., 2018), we deduce that pyoverdine’s importance in biocontrol activity is strongly dependent on the producer strain and the pathogen to be controlled. However, the limited role of pyoverdine in anti- Phytophthora activity of our two Pseudomonas strains does not mean that pyoverdine is not important for other functions in the strains. In multiple biological assays, we observed an increase in biocontrol activity when pyoverdine, or both pyoverdine and HCN were knocked out. Similarly to our previous findings that HCN is not only a toxin, but also a signaling molecule (Anand et al., 2023), there is evidence pointing to an indirect impact of the loss of pyoverdine on the R32’s overall biocontrol activity, potentially resulting from the upregulation of other biocontrol traits triggered by reduced iron concentrations in the bacteria. Iron is an important determinant of the whole regulatory machinery in bacteria (Hantke, 2001). Pyoverdine is not only used to acquire iron but is also believed to regulate iron homeostasis inside the cell (Schalk & Guillon, 2013). This implies that pyoverdine is indirectly involved in the regulatory network of Pseudomonas . This hypothesis is supported by the fact that pyoverdine has an important role in biofilm formation and swarming (Kang et al., 2018; Kang & Kirienko, 2017; Matilla et al., 2007). Matilla et al. (2007) even proposed that pyoverdine might be used in a kind of quorum-sensing mechanism in which iron would be the signal molecule and pyoverdine the regulator. In any case, the repeated observations that the lack of pyoverdine leads to higher antagonistic activity suggest that changes in iron sensing and homeostasis might be a major regulator of biocontrol activity in antagonistic Pseudomonas. The differing activities of our two strains and their respective mutants on the different developmental stages of P. infestans in vitro and in planta show that biocontrol is a complex phenomenon where the roles of specific molecules can be highly dependent on the producing strain, on the target pathogen and on the conditions of the interaction (e.g. taking place in laboratory media or on plant material). Moreover, it is important to keep in mind that the results obtained on leaf discs, though in planta , may not be representative of what would happen on whole plants, or even in field conditions. Understanding the true role of pyoverdine in whole plant and field assays would therefore be an important avenue to explore in future studies, as well as elucidating the mechanisms leading to increased biocontrol activity in strains deprived of pyoverdine. Conclusions In order to assess the role of pyoverdine in the antagonistic activity of two potato-associated Pseudomonas strains against. P. infestans , we created pyoverdine mutants in different genetic backgrounds and evaluated their performance in multiple developmental stages of the oomycete. We found that the contribution of pyoverdine strongly depended on the Pseudomonas strain, and that its lack often resulted in higher antagonistic activity. This finding is unexpected, given pyoverdine’s high affinity for iron and given the fact that P. infestans relies on iron supply for its growth and development (Cuppett & Lilly, 1973). Iron is also relevant to the host plant potato, to defend itself against the oomycete upon infection (Mata et al., 2001). All this shows the importance of iron and its acquisition for the pathogenesis of P. infestans and the protective activity of biocontrol strains. This brings us to the conclusion that more studies are needed to identify the mechanisms underlying the enhanced biocontrol activity observed in pyoverdine deficient mutants and to unravel how iron homeostasis regulates biocontrol-relevant traits in plant-associated Pseudomonas. Abbreviations ABC ATP-binding cassette CAS Chrome azurol S FUR Ferric uptake regulator HCN hydrogen cyanide KO Knock-out NRPS Non-ribosomal peptide synthetase Declarations Ethics approval Not applicable Consent for publication Not applicable Availability of data All referenced results can be found in results or supplementary data. Raw data can be provided upon request. Competing interests The authors declare that they have no competing interests. Funding Financial support from the Swiss National Science Foundation (grant nr. 207917 to LW) is gratefully acknowledged. Author’s contribution FLH created all the pvdE knock-out mutants and co-supervised the work together with LW. LJ performed all experiments, analyzed the data and wrote the first draft of the manuscript. All authors contributed to the writing and editing of the manuscript. LW secured the funding. Acknowledgments We thank Abhishek Anand for fruitful discussions and the sharing of his valuable expertise that helped in many aspects of the work. We also wish to thank Mout de Vrieze for her advice on the leaf discs assay, as well as Sébastien Bruisson and Rudolph Rohr for their guidance regarding the statistical analyses. References Anand, A., Chinchilla, D., Tan, C., Mène-Saffrané, L., L’Haridon, F., & Weisskopf, L. (2020). Contribution of hydrogen cyanide to the antagonistic activity of Pseudomonas strains against Phytophthora infestans. Microorganisms , 8 (8), 1–10. https://doi.org/10.3390/microorganisms8081144 Anand, A., Falquet, L., Abou-Mansour, E., L’Haridon, F., Keel, C., & Weisskopf, L. (2023). 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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-7139568","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":495984515,"identity":"fe858e9a-f747-44d7-bfad-cdc97e5f6d74","order_by":0,"name":"Livia Jerjen","email":"","orcid":"","institution":"University of Fribourg","correspondingAuthor":false,"prefix":"","firstName":"Livia","middleName":"","lastName":"Jerjen","suffix":""},{"id":495984516,"identity":"fa14e319-dd8d-4d45-907c-5980076e7418","order_by":1,"name":"Floriane L’Haridon","email":"","orcid":"","institution":"University of Fribourg","correspondingAuthor":false,"prefix":"","firstName":"Floriane","middleName":"","lastName":"L’Haridon","suffix":""},{"id":495984517,"identity":"c3a4275f-6b24-40ae-9373-b63c0fe0c6fc","order_by":2,"name":"Laure Weisskopf","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIie3QMYvCMBTA8VcKdem1a6f2K6QEHP0sDQW7VBeXbpcg1EVwtd+i4OKodHDpByjURQSnG3qbg+C9u1MEMdrRIf/lhcAPXgKgUr1h1vVgAOg4XBdA4w2eAhkx7gilSMS8FYELYRzNc9Ipu00DhWvZmz35XpIor8dinEBvKCVmTLM5FNRwYsqykgzy7VqIEsKRfLGY6ibULHVMvfhIT4O8YpMdh9Xvho+J/UX1E9SfKS6GhESkYkI8JbgPPrsODAhoiCRoQQ4jbUrOforWx7f42R8hoZTYdriAY9L3vNlm7+CPeVYV7QRPelLyH7lMjd/fvOxGVCqVSnXrB22mViIsuj5KAAAAAElFTkSuQmCC","orcid":"","institution":"University of Fribourg","correspondingAuthor":true,"prefix":"","firstName":"Laure","middleName":"","lastName":"Weisskopf","suffix":""}],"badges":[],"createdAt":"2025-07-16 11:38:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7139568/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7139568/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88530128,"identity":"ff3b717b-a8f0-422e-a53a-679124f7f66f","added_by":"auto","created_at":"2025-08-07 11:20:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10444349,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSiderophore detection in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePseudomonas\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e R32 and R47.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e: Pyoverdine levels were measured by fluorescence emission in 48 h old bacterial cultures grown in KB. Negative control values were subtracted from sample values first, after which sample values were normalized to OD\u003csub\u003e600\u003c/sub\u003e=1. Bars represent the mean of 2 biological replicates with each 3 technical replicates. Statistical analysis was performed using a one-way ANOVA, followed by a Tukey’s test. \u003cstrong\u003eB: \u003c/strong\u003ePictures of the siderophore detection assays on plates. On the left side of each plate, bacteria were grown on KB medium for 48 h and then pictures were taken under UV light in order to visualize fluorescent molecules (mainly fluorescent siderophores). On the right side of the plates, bacteria were grown on KB-CAS medium for 48 h. Siderophore diffusion is visualized by an orange halo. Pictures shown are representative examples of 2 technical replicates.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7139568/v1/899649ccbb614e52cf94193b.png"},{"id":88531195,"identity":"245310f7-f605-4362-b27c-38cd08a238f3","added_by":"auto","created_at":"2025-08-07 11:36:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":33718336,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePyoverdine detection in Pseudomonas R32 and R47.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e Pyoverdine levels were measured by fluorescence emission in 48 h old bacterial cultures grown in KB. Negative control values were subtracted from sample values first and then normalized to OD\u003csub\u003e600\u003c/sub\u003e=1. Bars represent the mean of 2 biological replicates with each 3 technical replicates. See Table S3 for statistical analysis. \u003cstrong\u003eB: \u003c/strong\u003ePictures of pyoverdine fluorescence on plates. Bacteria were grown on KB medium for 48 h. On the left side, pictures under normal light were taken. On the right side, we can see the exact same cultures, but pictures were taken under UV light in order to visualize fluorescent molecules, mainly fluorescent siderophores.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7139568/v1/c045425f92919a6e9d855725.png"},{"id":88530998,"identity":"be739433-b28d-44c3-bc78-257f57fd9814","added_by":"auto","created_at":"2025-08-07 11:28:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":29507399,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMycelial inhibition assay on media with different iron concentrations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. infestans \u003c/em\u003eand different genotypes of \u003cem\u003ePseudomonas \u003c/em\u003eR32 and R47 were grown for 14 days on V8 medium supplemented with different FeCl\u003csub\u003e3 \u003c/sub\u003econcentrations (0 mg.L\u003csup\u003e-1\u003c/sup\u003e on the left\u003cem\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003c/em\u003eincreasing concentrations to the right). \u003cstrong\u003eA:\u003c/strong\u003e The mean inhibition percentage was calculated relative to the negative control, which represents 0 % of inhibition. Averages of 2 biological replicates with each 4 technical replicates with standard deviation are shown. See Table S4 for statistical analysis. \u003cstrong\u003eB:\u003c/strong\u003e Representative pictures of the inhibition assays that were used to generate the data in A.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7139568/v1/145f1eed667bb0b6fd6119cc.png"},{"id":88530139,"identity":"65769b2b-6dc3-4bc8-bf0e-97fa8144ac20","added_by":"auto","created_at":"2025-08-07 11:20:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":13893748,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eP. infestans \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ezoospore release in presence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePseudomonas\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e R32 and R47.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA and B: \u003c/strong\u003ePercentages of empty sporangia of \u003cem\u003eP. infestans\u003c/em\u003e GFP strain exposed to bacteria at OD\u003csub\u003e600\u003c/sub\u003e=0.25 (\u003cstrong\u003eA\u003c/strong\u003e) or OD\u003csub\u003e600\u003c/sub\u003e=0.5 (\u003cstrong\u003eB\u003c/strong\u003e). Bars represent the mean of 3 biological replicates with each 3 technical replicates. Statistical analysis was performed using a Kruskal-Wallis multiple comparisons test, followed by a Dunn’s test. \u003cstrong\u003eC: \u003c/strong\u003eRepresentative pictures of the assay at OD\u003csub\u003e600\u003c/sub\u003e=0.25. Zoomed cut-outs are shown.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7139568/v1/abc05b0f3684be4aedfa1948.png"},{"id":88530137,"identity":"5996c9ca-6f8e-4908-92f9-f318f084f6b4","added_by":"auto","created_at":"2025-08-07 11:20:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":14862360,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eP. infestans \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ezoospore germination in presence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePseudomonas\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e R32 and R47.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA and B: \u003c/strong\u003ePercentages of germinated zoospores of \u003cem\u003eP. infestans\u003c/em\u003e GFP strain exposed to bacteria at OD\u003csub\u003e600\u003c/sub\u003e=0.25 (\u003cstrong\u003eA\u003c/strong\u003e) or OD\u003csub\u003e600\u003c/sub\u003e=0.5 (\u003cstrong\u003eB\u003c/strong\u003e). Bars represent the mean of 3 biological replicates with each 3 technical replicates. Statistical analysis was performed using a Kruskal-Wallis multiple comparisons test, followed by a Dunn’s test. \u003cstrong\u003eC: \u003c/strong\u003eRepresentative pictures of the assay at OD\u003csub\u003e600\u003c/sub\u003e=0.25. Zoomed cut-outs are shown.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7139568/v1/b8a74769f64805839676c374.png"},{"id":88531194,"identity":"456e5f30-adfe-4fc8-ae1a-96744aa52435","added_by":"auto","created_at":"2025-08-07 11:36:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":16176893,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eP. infestans \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eGFP\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003esporangia germination in presence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePseudomonas\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e R32 and R47.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBars represent the mean of 2 biological replicates with each 3 technical replicates. Statistical analysis for germ tube length was performed with a one-way ANOVA followed by a Tukey’s test. For percentage of germinated sporangia, we used a Kruskal-Wallis multiple comparisons test, followed by a Dunn’s test. \u003cstrong\u003eA: \u003c/strong\u003ePercentages of germinated sporangia exposed to R32 at OD\u003csub\u003e600\u003c/sub\u003e=0.01. \u003cstrong\u003eB\u003c/strong\u003e: Percentages of germinated sporangia exposed to R32 at OD\u003csub\u003e600\u003c/sub\u003e=0.05. \u003cstrong\u003eC: \u003c/strong\u003eThe length of the germ tubes exposed to R32 at OD\u003csub\u003e600\u003c/sub\u003e=0.01 was measured in pixels. \u003cstrong\u003eD\u003c/strong\u003e: Percentages of germinated sporangia exposed to R47 at OD\u003csub\u003e600\u003c/sub\u003e=0.25. \u003cstrong\u003eE:\u003c/strong\u003e Percentages of germinated sporangia exposed to R47 at OD\u003csub\u003e600\u003c/sub\u003e=0.5. \u003cstrong\u003eF:\u003c/strong\u003e The length of the germ tubes exposed to R47 at OD\u003csub\u003e600\u003c/sub\u003e=0.25 was measured in pixels. \u003cstrong\u003eG\u003c/strong\u003e: Representative pictures of the assays at OD\u003csub\u003e600\u003c/sub\u003e=0.01 for R32 and OD\u003csub\u003e600\u003c/sub\u003e=0.25 for R47 genotypes. Zoomed cut-outs are shown.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-7139568/v1/1a4a07ba1d8b7449af96e751.png"},{"id":88530989,"identity":"a5ce1afe-fd47-4618-bc56-2a58e0eea8cc","added_by":"auto","created_at":"2025-08-07 11:28:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":12851743,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLeaf disc infection assay in presence of R32 and R47.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLeaf discs from the Bintje cultivar were infected with \u003cem\u003eP. infestans\u003c/em\u003eGFP zoospores and at the same time inoculated with different genotypes of R32 and R47. Each treatment was tested on 20 individual leaf discs. \u003cstrong\u003eA: \u003c/strong\u003eLesion formation was scored from 1 (no lesion) to 5 (full necrosis) at 4 dpi. Statistical analysis was performed with a Kruskal-Wallis test followed by a Dunn’s test. \u003cstrong\u003eB:\u003c/strong\u003e Mycelium formation was scored from 1 (no mycelium) to 5 (extensive myelium) at 5 dpi. The same statistical analysis as for lesion score was performed. \u003cstrong\u003eC:\u003c/strong\u003e Pictures from one representative leaf disc per treatment.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-7139568/v1/3e852275af9ae2d0311032d4.png"},{"id":90147714,"identity":"c7d359f1-4ebf-4937-ad13-ec10499a4679","added_by":"auto","created_at":"2025-08-29 06:19:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":122696546,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7139568/v1/5df45f0e-ca81-4329-ac0a-d5a62c882003.pdf"},{"id":88530125,"identity":"eba0f9ac-b0a5-45d5-b03c-71d6009e1719","added_by":"auto","created_at":"2025-08-07 11:20:43","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":33594,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-7139568/v1/b5c973c17f15cd150b08a308.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluating the contribution of pyoverdine to the anti-Phytophthora activity of two potato-associated Pseudomonas strains","fulltext":[{"header":"Background","content":"\u003cp\u003eCrops are exposed to a variety of pathogens that can cause severe yield losses. In potato in particular, \u003cem\u003ePhytophthora infestans\u003c/em\u003e stands out as a challenging pathogen worldwide (Kamoun et al., 2015). This oomycete, causing late blight in \u003cem\u003eSolanaceae\u003c/em\u003e crops, can destroy a potato field in 10\u0026ndash;15 days only (Rakotonindraina et al., 2012). It is considered the most threatening potato pathogen due to its rapid evolution, high adaptation capacity and fast asexual reproduction cycle involving two types of spores; sporangia that germinate directly under warmer conditions, and motile zoospores that are released from sporangia when temperatures are colder (Fry et al., 2015). Current crop protection practices heavily rely on copper-based and synthetic pesticides, the latter being not only toxic but also prone to resistance development in such a rapidly evolving pathogen as \u003cem\u003eP. infestans\u003c/em\u003e (Leesutthiphonchai et al., 2018). In contrast, biocontrol agents are less likely to trigger resistance development in pathogens due to their multi-target modes of action (Ivanov et al., 2021). In bacteria, these modes of action can encompass induction of plant resistance, or more direct mechanisms like antibiosis and competition for resources and space (Wang \u0026amp; Long, 2023). The latter includes also the competition for iron, which despite its abundance in the environment, is difficult to acquire in soils due to its insolubility in the ferric state (Owen \u0026amp; Ackerley, 2011). Microorganisms are able to produce iron-scavenging molecules, called siderophores, that allow them to take up the insoluble ion from the soil and make them great competitors for iron (Schalk, 2008), a feature that has been shown to play an important role in biocontrol of plant pathogens (Liu et al., 2021; Ran et al., 2005).\u003c/p\u003e\u003cp\u003ePrevious research in our group has focused on two \u003cem\u003ePseudomonas\u003c/em\u003e strains showing promise for the biological control of late blight: \u003cem\u003ePseudomonas sp.\u003c/em\u003e R32, which belongs to the \u003cem\u003eP. putida\u003c/em\u003e group, and \u003cem\u003ePseudomonas sp.\u003c/em\u003e R47, which belongs to the \u003cem\u003eP. protegens\u003c/em\u003e group. Both strains showed strong inhibitory activity on different developmental stages of \u003cem\u003eP. infestans\u003c/em\u003e (mycelium, sporangia, zoospores) (Anand et al. 2020, 2023; Hunziker et al. 2015). A comparative genomics approach identified putative biocontrol traits and mechanisms in these two strains (De Vrieze et al., 2018, 2020; Hunziker et al., 2015), among which the production of phenazines by \u003cem\u003ePseudomonas sp.\u003c/em\u003e R47, and the production of hydrogen cyanide (HCN) and pyoverdine by both strains. The role of hydrogen cyanide in both strains\u0026rsquo; anti-\u003cem\u003ePhytophthora\u003c/em\u003e activity has been investigated previously (Anand et al., 2020), and was shown to affect mycelial growth but not spore germination, nor disease progression in leaf discs. In contrast, the importance of pyoverdine for the anti-\u003cem\u003ePhytophthora\u003c/em\u003e activity has not been evaluated yet.\u003c/p\u003e\u003cp\u003ePyoverdine was first described as a siderophore in 1978 (Meyer \u0026amp; Hornsperger, 1978). Since then, its role in iron competition has been well established (Schalk \u0026amp; Guillon, 2013; Visca et al., 2007) and its synthesis and regulation are also well-studied (Henr\u0026iacute;quez et al., 2019; Ringel \u0026amp; Br\u0026uuml;ser, 2018; Yeterian et al., 2010). All pyoverdines are composed of a fluorescent dihydroquinoline-type chromophore which is linked to a strain-specific peptide chain and a side chain at the C3 position (Schalk \u0026amp; Guillon, 2013). Pyoverdine\u0026rsquo;s complex synthesis is regulated at multiple levels; by the global two-component system regulator GacS/GacA, directly by intracellular iron concentrations through the ferric uptake regulator (FUR) (Ochsner et al., 1995), by several transcriptional LysR-type regulators (Kang \u0026amp; Kirienko, 2017) and at post-transcriptional level by FpvR (Edgar et al., 2014). The synthesis involves a total of twelve non-ribosomal peptide synthetases (NRPS) and other proteins (Visca et al., 2007).\u003c/p\u003e\u003cp\u003eAlthough siderophores in general, and pyoverdine in particular, have been shown to play a role in the biological control of plant diseases (Liu et al., 2021; Ran et al., 2005), the extent of pyoverdine\u0026rsquo;s contribution to the anti-\u003cem\u003ePhytophthora\u003c/em\u003e properties of \u003cem\u003ePseudomonas\u003c/em\u003e strains remains unknown. To fill this gap of knowledge, we created pyoverdine mutants in both wild-types and HCN-deletion mutants of the two \u003cem\u003ePseudomonas\u003c/em\u003e biocontrol strains R32 and R47. We selected \u003cem\u003ePvdE\u003c/em\u003e, which encodes an ATP-binding cassette (ABC) transporter that exports pyoverdine\u0026rsquo;s precursor ferribactin from the cytosol into the periplasm for the maturation process, as a target for mutagenesis, since knocking-out this gene has been reported to abolish pyoverdine production in \u003cem\u003eP. aeruginosa\u003c/em\u003e (Yeterian et al., 2010).\u003c/p\u003e\u003cp\u003eWe characterized the ability of these mutants to inhibit different developmental stages of \u003cem\u003eP. infestans\u003c/em\u003e in \u003cem\u003ein vitro\u003c/em\u003e experiments, as well as their impact on disease progression \u003cem\u003ein planta\u003c/em\u003e, to gain a detailed understanding of pyoverdine\u0026rsquo;s putative role in the inhibition of the causative agent of late blight by two different potato-associated \u003cem\u003ePseudomonas\u003c/em\u003e strains.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cb\u003eStrains and culture conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003ePseudomonas\u003c/em\u003e sp. R32 and \u003cem\u003ePseudomonas\u003c/em\u003e sp. R47 were originally isolated from the rhizosphere of potato plants (Hunziker et al., 2015). The two wild-types and their respective mutants were routinely cultured on LB-Agar plates with 10 \u0026micro;M rifampicin at 28\u0026deg;C. LB agar plates were prepared with 12.5 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e LB Broth Miller (Roth), 10 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e LB Broth Lennox (Roth) and 15 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of Agar-Agar-Kobe I (Roth), which were dissolved in distilled water and autoclaved. Liquid cultures were incubated overnight at 180 rpm at 28\u0026deg;C.\u003c/p\u003e\u003cp\u003eKB medium was prepared by mixing 20 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e proteose peptone #3 (Gibco), 1.5 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e (Roth), 1.5g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MgSO\u003csub\u003e4\u003c/sub\u003e x 7H\u003csub\u003e2\u003c/sub\u003e0 (Roth), 10 ml.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Glycerol (Reactolab) and 15 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Agar-agar Kobe I (Roth) for solid medium, dissolving them in distilled water and autoclaving.\u003cem\u003eP. infestans\u003c/em\u003e strains Rec01, 44 and 208m2 (GFP-tagged strain, (Si-Ammour, Mauch-Mani, and Mauch 2003)) were routinely grown at 18\u0026deg;C on V8-Agar plates. This medium was prepared by diluting V8 100 % hot spicy vegetable juice at 100 ml.L\u003csup\u003e1\u003c/sup\u003e in distilled water, and adding of 1 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of CaCO\u003csub\u003e3\u003c/sub\u003e (Roth) and 15 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of agar. Liquid V8 medium was filtered in some experiments with 0.22 \u0026micro;m filters (Millex) after autoclaving in order to get rid of debris. After a maximum of seven passages on plates, \u003cem\u003eP. infestans\u003c/em\u003e was passaged on potato tubers to keep its virulence.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGeneration of knock-out (KO) mutants in\u003c/b\u003e \u003cb\u003ePseudomonas\u003c/b\u003e \u003cb\u003eR32 and R47\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe followed the same protocol as described earlier (Anand et al., 2020) to generate \u003cem\u003epvdE\u003c/em\u003e KO mutants in both R32 and R47 wild-type and ∆\u003cem\u003ehcn\u003c/em\u003e strains. Since for R32, the attempts to amplify the gene fragments for the first cloning step were unsuccessful, these sequences were produced synthetically by Eurofins (\u0026copy; Eurofins Scientific 2023). Please refer to Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e for the list of plasmids and strains, and Table S2 for the primers used and generated.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGrowth curves and monitoring of pyoverdine production over time\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOvernight bacterial cultures in LB were centrifuged at 5000 rpm, the supernatant removed, and the pellet resuspended in 0.9% NaCl (Roth). After a second centrifugation step, the washing supernatant was removed and resuspended in the same solution. OD\u003csub\u003e600\u003c/sub\u003e was adjusted to 1 and in a 96-well plate, we mixed 5 \u0026micro;L of the bacteria (or 0.9% NaCl for the negative control) with 195 \u0026micro;L of either liquid LB, KB or filtered V8. We performed triplicates for each sample. Then the plate was incubated during 60 h in the Cytation 5 with shaking at 28\u0026deg;C. Every hour, absorbance at 600 nm and pyoverdine fluorescence (excitation at 405 nm, emission at 460 nm) were measured.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSiderophore detection with Chrome azurol S (CAS)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCAS media were prepared by mixing 10 mL of iron solution (270 mg.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FeCl\u003csub\u003e3\u003c/sub\u003e x 6 H2O (Merck) and 1% HCl 1M (Fisher Chemicals) in distilled water) with 50 mL of solution 1 (1.21 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e chrome azurol S (Kodak) in distilled water). 40 mL of solution 2 (1,8225 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e HDTMA (Sigma) dissolved in distilled water) were added to the first mix, and the final CAS solution was autoclaved. KB-Agar was prepared as described above and before autoclaving, 30.24 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of PIPES PUFFERAN (Roth) were added, the pH was adjusted to 6.8 with NaOH and then 15 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of agar were added. KB-medium was autoclaved, then mixed with the CAS solution in a ratio of 9:1. Overnight bacterial cultures in LB were centrifuged at 5000 rpm, the supernatant removed, and the pellet resuspended in 0.9% NaCl (Roth). After a second centrifugation step, the washing supernatant was removed and resuspended in the same solution. OD\u003csub\u003e600\u003c/sub\u003e was adjusted to 1 and 10 \u0026micro;L of the bacterial suspensions were pipetted onto CAS-KB plates before incubating for 48 h at 28 \u0026deg; C.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePyoverdine measurements\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOvernight cultures of bacteria in LB were rinsed in 0.9% NaCl (Roth) as described above and adjusted to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1 in the same solution. In triplicates, we added 5 \u0026micro;L of bacterial suspension (or 0.9% NaCl for the negative control) to 195 \u0026micro;L KB or KB supplemented with FeCl\u003csub\u003e3\u003c/sub\u003e solution (see CAS iron solution above) in 96-well plates (Costar). Parafilmed plates were incubated for 48 h at 28\u0026deg;C under shaking at 120 rpm. Pyoverdine fluorescence (excitation at 405 nm, emission at 460 nm) and OD\u003csub\u003e600\u003c/sub\u003e were measured in a Cytation 5 cell imaging reader (Biotek). Both the negative controls of fluorescence and OD values were subtracted from the measured sample values, and those subsequently normalized to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1. After normalization, mean values and SD were calculated for each sample and statistical analysis was performed using a one-way ANOVA, followed by a Tukey\u0026rsquo;s test, where \u0026lowast;=p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u0026lowast;\u0026lowast;=p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and \u0026lowast;\u0026lowast;\u0026lowast;=p\u0026thinsp;\u0026lt;\u0026thinsp;0.001. For the assay with FeCl\u003csub\u003e3\u003c/sub\u003e supplementation, we performed a mixed-effect model analysis with Geisser-Greenhouse correction, followed by a Tukey\u0026rsquo;s test.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHCN detection\u003c/b\u003e\u003c/p\u003e\u003cp\u003e50 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of copper(II)-ethyl acetoacetate (Aldrich) and 50 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 4,4-methylenbis(N,N-dimethylaniline) (Sigma-Aldrich) were dissolved in chloroform (Fisher Chemicals). This solution was then pipetted onto pieces of filter paper (Whatman) that had been cut beforehand. The paper was dried overnight under a fume hood and then stored in a glass container wrapped in aluminum foil at 4\u0026deg;C. Split Petri dishes (Sarstedt) were filled on one side with LB-Agar and left empty on the other side. 10 \u0026micro;L of bacteria at OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1 were pipetted onto the LB-Agar and a detection paper placed into the empty dish side. Plates were sealed with parafilm and incubated at 30\u0026deg;C, pictures were taken at 24 h and 48 h after incubation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003edual assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003eExperiments were performed on plates filled with V8-Agar, which in some experiments was supplemented with FeCl\u003csub\u003e3\u003c/sub\u003e (see CAS iron solution above). Overnight cultures of bacteria in LB were rinsed in 0.9% NaCl (Roth) as described above and adjusted to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1 in the same solution. Three drops of 10 \u0026micro;L of bacterial suspension (or 0.9% NaCl for negative control) were carefully applied symmetrically onto an V8-Agar plate near the border. A plug of \u003cem\u003eP. infestans\u003c/em\u003e Rec01 culture was then placed in the middle of the plate. Sealed plates were incubated at 21\u0026deg;C for 12\u0026ndash;14 days. Four replicate plates per bacterial strain and six replicate plates for the control (\u003cem\u003eP. infestans\u003c/em\u003e growing alone) were performed per experiment. For the analysis, the area of growth of the pathogen\u0026rsquo;s mycelium and the area of the bacterial colony were measured with ImageJ (Schneider et al., 2012). The percentage of growth inhibition caused by the bacteria was calculated with the following formula: \u0026#119868;\u0026#119899;ℎ\u0026#119894;\u0026#119887;\u0026#119894;\u0026#119905;\u0026#119894;\u0026#119900;\u0026#119899; %=100\u0026lowast;\u0026#119860;\u0026#119894;/(\u0026#119860;\u0026#119862;\u0026minus;\u0026#119860;\u0026#119887;), with inhibition area \u0026#119860;\u0026#119894; = \u0026#119860;\u0026#119862;\u0026minus;\u0026#119860;\u0026#119887;\u0026minus;\u0026#119860;\u0026#119905;, where \u0026#119860;\u0026#119862;= pathogen area in control plates, \u0026#119860;\u0026#119887;= bacterial area, \u0026#119860;\u0026#119905;= pathogen area. Note that the inhibition area was calculated considering that the bacterial area is not available anymore for the pathogen because of space competition (\u0026#119860;\u0026#119862;\u0026minus;\u0026#119860;\u0026#119887;). We performed a mixed-effect model analysis with Geisser-Greenhouse correction, followed by a Tukey\u0026rsquo;s test, where \u0026lowast;=p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u0026lowast;\u0026lowast;=p \u0026lt; 0.01, and \u0026lowast;\u0026lowast;\u0026lowast;=p \u0026lt; 0.001.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSpore assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe performed zoospore release, zoospore germination and sporangia germination assays, and for all three types, an overnight liquid culture of bacteria in LB was rinsed in 0.9% NaCl (Roth) as described above and adjusted to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1 in filtered V8. 50 \u0026micro;L of bacterial suspension were transferred into flat-bottom 96-well plates (Costar). The procedure for each assay differed from here, as detailed below.\u003c/p\u003e\u003cp\u003eFor the zoospore germination assay, 50 \u0026micro;L of 0.9% NaCl were added to the bacterial suspension. Then, 10 mL ice cold, sterile water were poured onto 2 to 3 week-old \u003cem\u003eP. infestans\u003c/em\u003e cultures on V8-agar plates which were placed at 4\u0026deg;C in the dark for 2 h. After this cold shock, the plates were placed at 18\u0026deg;C for 30 min. Zoospores were then collected by aspiring the water from the mycelium surface with a pipette and their concentration adapted to 3x10\u003csup\u003e5\u003c/sup\u003e zoospores.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. 40 \u0026micro;L of zoospore solution were added into the wells. The sealed plates were then incubated in the dark at 18\u0026deg;C for 3\u0026ndash;4 h.\u003c/p\u003e\u003cp\u003eFor the zoospore release assay, sporangia were collected by adding 2\u0026ndash;3 mL of sterile water onto a 2 to 3 week-old \u003cem\u003eP. infestans\u003c/em\u003e plate and scratching off the mycelium from the agar with a glass slide. The mycelium was washed through a filter with sterile water into a 15 mL falcon tube (Cellstar), so that sporangia were separated from the mycelium. The sporangia solution was then adapted to 3x10\u003csup\u003e5\u003c/sup\u003e sporangia.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 40 \u0026micro;L added into wells. 50 \u0026micro;L of ice cold sterile water were added into the wells and the plate was placed at 4\u0026deg;C in the dark for 2 h, then for 30 min at 18\u0026deg;C, still in the dark.\u003c/p\u003e\u003cp\u003eFor the sporangia germination assays, 50 \u0026micro;L of 0.9% NaCl were added to the wells. Sporangia were collected as described above and 40 \u0026micro;L of the final solution of 3x10\u003csup\u003e5\u003c/sup\u003e sporangia.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was added before incubating in humid, dark conditions for 24 h.\u003c/p\u003e\u003cp\u003eAfter the respective incubation times, pictures were taken with the Cytation 5 (Biotek), and then percentages of germination for sporangia and zoospores were calculated by counting germinated vs. non-germinated spores (total spores per replicate ca. 200 spores), and release percentage was calculated by counting full vs. empty sporangia, counting the same number of spores as above per picture. All experiments were performed in technical triplicates. For statistical analysis, we pooled together results from 4 experiments and used a Kruskal-Wallis test followed by a Dunn\u0026rsquo;s multiple comparisons test performed either on GraphPad Prism or R Studio.\u003c/p\u003e\u003cp\u003eFor sporangia germination, we measured additionally the length of germ tubes with ImageJ in 2 experiments with each three technical replicates and performed an ANOVA with Tukey\u0026rsquo;s post-hoc test for statistical analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLeaf disc assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePotato plants from the Bintje cultivar were cultivated in a greenhouse for 6\u0026ndash;7 weeks. Leaf discs were cut and placed abaxial side up onto a 0.8% water agar plate (5 discs per plate) the day before inoculation and infection. We analyzed 4 plates per bacterial treatment and negative control (20 leaf discs). One leaf disc per plant was used as a non-infected control. Bacteria were cultured overnight in LB, washed in 0.9% NaCl and resuspended at OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2 in the same solution. Zoospores were harvested from 2 week-old \u003cem\u003eP. infestans\u003c/em\u003e cultures as described above. The zoospore solution was adjusted to 6-8x10\u003csup\u003e5\u003c/sup\u003e spores.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The bacterial suspension and zoospore solution were mixed in a 1:1 ratio and 10 \u0026micro;L of this mixture was applied onto each leaf disc. Plates were then placed into a transparent, closed box with high humidity at 21\u0026deg;C with a light/dark cycle for max. 6 days. Pictures were taken at 4 dpi and 5 dpi. Lesion development was scored at 4 dpi from 0 (no lesion) to 5 (full necrosis) and mycelium development was scored at 5 dpi from 0 (no mycelium) to 5 (fully covered by mycelium). We used a Kruskal-Wallis test followed by a Dunn\u0026rsquo;s multiple comparisons test for both scores separately.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eDeletion of\u003c/b\u003e \u003cb\u003epvdE\u003c/b\u003e \u003cb\u003eleads to decreased secretion of pyoverdine in both R32 and R47\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the impact of siderophore production on the anti-\u003cem\u003ePhytophthora\u003c/em\u003e activity of \u003cem\u003ePseudomonas\u003c/em\u003e R32 and R47, pyoverdine mutants were generated by deleting the \u003cem\u003epvdE\u003c/em\u003e gene in both wild-type and HCN-deletion mutant backgrounds (Anand et al., 2020). This mutation did not impair the strains\u0026rsquo; growth (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). As expected, pyoverdine levels as revealed by fluorescence emission (Kang \u0026amp; Kirienko, 2017) were strongly reduced in single and double mutants of both strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Fig. S2), although production was not completely abolished in R32. Higher pyoverdine emission was observed in R32 than in R47 and in single ∆\u003cem\u003ehcn\u003c/em\u003e mutants than in the respective wild-type strains, as previously reported (Anand et al., 2023). This was visible in the pyoverdine measurement in liquid cultures of R32 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and in the UV fluorescence emitted on solid culture plates for R47 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). To gain a broader view of siderophore production beyond the measurement and visualization of fluorescent molecules, we grew the eight genotypes on CAS plates, on which siderophore-mediated iron depletion can be visualized by an orange halo. In contrast to our expectations, we observed no clear correlation between pyoverdine fluorescence measurements and the size of the orange halo, which suggests that this halo is mediated by the secretion of other siderophores.\u003c/p\u003e\u003cp\u003eAs expected, pyoverdine production correlated with iron deficiency, although differences were observed between the two strains: R32 gradually decreased its pyoverdine production with increasing iron concentrations but still produced detectable amounts at the highest iron supply tested (27 mg.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), while R47 stopped producing pyoverdine already when supplied with 1 mg.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e iron in its growth medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIron supply decreased the ability to restrict\u003c/b\u003e \u003cb\u003eP. infestans\u003c/b\u003e \u003cb\u003emycelial growth in R32, but not in R47\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn V8 medium without iron supplementation, we observed no significant loss of activity in single ∆\u003cem\u003epvdE\u003c/em\u003e mutants compared to the wild-type strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B, Table S4). While all R47 mutants kept the full inhibition potential of the wild-type, slight modulations were observed in R32, with a small but significant decrease of activity in the single ∆\u003cem\u003ehcn\u003c/em\u003e mutant and a partial rescue of the wild-type phenotype in the double mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Table S4). Adding increasing concentrations of iron did not impair R47\u0026rsquo;s ability to inhibit mycelial growth, suggesting that competition for iron is not involved in this process. In contrast, both wild-type and mutant strains of R32 lost part of their activity with increasing iron supply, suggesting that part of the observed inhibition potential was due to iron deprivation of \u003cem\u003eP. infestans\u003c/em\u003e. This was most visible in the absence of HCN. However, ∆\u003cem\u003epvdE\u003c/em\u003e mutants did not show a more severe activity loss than their corresponding controls (wild-type vs. single ∆\u003cem\u003ehcn\u003c/em\u003e mutant) as could have been expected if pyoverdine had played a role in iron acquisition. On the contrary, the double knock-out seemed to regain part of the wild-type\u0026rsquo;s inhibitory potential independently of iron supplementation, suggesting the putative upregulation of a yet unknown factor leading to mycelial restriction when both HCN and pyoverdine are no longer produced.\u003c/p\u003e\u003cp\u003eIt was striking to observe that R32 and its respective mutants were accumulating and secreting a reddish molecule when growing on plates supplemented with high iron concentrations, a phenomenon that was the most intense in the single HCN mutant in R32. In R47, no secretion of this molecule could be observed, only a slightly darker coloration in the HCN mutant and double mutant at the highest iron concentrations. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). A phenotype visible in both strains was the decreasing colony diameter with increasing iron concentrations which could be due to a higher agar density mediated by iron addition, and was not linked to toxicity, as cell numbers revealed by CFU counting were not reduced even at the highest iron dose (Fig. S3).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMutants deprived of both HCN and pyoverdine lost their spore inhibition potential in R32, but not in R47.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn addition to mycelial growth inhibition, we analyzed whether pyoverdine would play a role in the inhibition of zoospore release (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), zoospore germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) and sporangia germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). As spore physiology shows higher variability between pathogen strains than mycelial growth, we analyzed three different \u003cem\u003eP. infestans\u003c/em\u003e genotypes. Similar results were obtained for the three strains (see Fig. S4, S5 and S6), hence we only present the results obtained with the GFP-labeled strain in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. In general, R47 showed very little spore inhibition potential compared to R32, which led to strong inhibition even when applied at low cell densities. In both zoospore release and germination, no significant change of activity was observed in the mutant genotypes of R47 compared to the wild-type at both tested concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Although not significant, we could still observe a tendency towards increased antagonistic activity for the R47 HCN single mutant and the double mutant in the zoospore germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). In R32, both single mutants kept their full potential and a strong loss of inhibition was observed only for the double mutant. For zoospore release, the effect that was only a tendency at lower bacterial concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) became a significant loss of inhibition at the higher concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), where it almost reached the same level of release as in the water control. Regarding the zoospore germination, at both tested concentrations, the difference between the double mutant and all other R32 genotypes was not statistically significant due to high variability between replicates, but a clear trend was visible (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). Since no loss of inhibition was detected for either of the single mutants, this result suggests an interplay or a redundant effect of both biocontrol traits.\u003c/p\u003e\u003cp\u003eSporangia germination yielded a contrasting picture, in which no difference between wild-type and mutants was observed neither in R32 nor in R47 for germination percentages (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B, D and E). When looking at germ tube length, neither pyoverdine nor HCN loss affected the inhibition ability of R32 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), while for R47, both single and double ∆\u003cem\u003ehcn\u003c/em\u003e mutants showed higher activity than the wild-type, suggesting the upregulation of other active compounds in absence of HCN (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDual loss of HCN and pyoverdine decreased\u003c/b\u003e \u003cb\u003ein planta\u003c/b\u003e \u003cb\u003eactivity in R47 but increased activity in R32\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSince infection of the leaf discs was performed with zoospores in direct contact with the bacteria, we expected to have similar results as in the zoospore germination assays. Surprisingly, the results showed opposite trends compared to the \u003cem\u003ein vitro\u003c/em\u003e experiment, underlining the differing conditions of \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein planta\u003c/em\u003e assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In R32, the \u003cem\u003epvdE\u003c/em\u003e single mutant displayed a significant increase of protection against \u003cem\u003eP. infestans\u003c/em\u003e compared to the wild-type, both when looking at lesion size (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) or mycelium development (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The double mutant also was significantly more protective compared to the wild-type, but not when compared to ∆\u003cem\u003ehcn\u003c/em\u003e, since this mutant already showed a trend towards higher protection. These findings suggest an upregulation of one or several other biocontrol traits in absence of HCN and pyoverdine. In the case of R47, we observed a complete loss of protective activity in all mutant strains compared to the wild-type. In this case, HCN and pyoverdine appeared to be important traits to protect leaf discs against \u003cem\u003eP. infestans\u003c/em\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo assess the importance of pyoverdine in the biocontrol activity of two \u003cem\u003ePseudomonas\u003c/em\u003e strains against \u003cem\u003eP. infestans\u003c/em\u003e, we knocked out \u003cem\u003epvdE\u003c/em\u003e in both wild-type and HCN-deletion mutants. Although Yeterian et al. (2010) reported to prevent pyoverdine production and accumulation in \u003cem\u003eP. aeruginosa\u003c/em\u003e by knocking out this gene, we could only achieve the same in one of our two strains. In R32, both the single \u003cem\u003epvdE\u003c/em\u003e mutant and the double mutant still displayed a decreased but measurable pyoverdine level, corresponding approximately to the wild-type level in R47 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). While it was still significantly reduced compared to the wild-type in R32, the upcoming question is why we could not achieve a complete depletion of pyoverdine in this strain. Although we cannot exclude a spontaneous diffusion through the membrane, we looked for homologues in the genome of R32 which could take over the transport of ferribactin into the periplasm, but without conclusive results. Although the levels measured in the mutant of R32 might still be biologically relevant, we decided to go further with this mutant, with the hypothesis that a 7-fold reduction in pyoverdine levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) would be enough to assess the relevance of pyoverdine in the strain\u0026rsquo;s anti-\u003cem\u003ePhytophthora\u003c/em\u003e activity.\u003c/p\u003e\u003cp\u003eWhen measuring pyoverdine-specific fluorescence levels, we could clearly observe higher amounts of pyoverdine in R32 than in R47 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In contrast, in the fluorescence pictures we can see a more pronounced intensity in R47 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Under broad spectrum UV other fluorescent molecules can be visible, which explains the observed discrepancy. The more intense fluorescence for R47 \u003cem\u003e∆hcn\u003c/em\u003e compared to R47 WT could correspond to another fluorescent siderophore, which would be activated by pyoverdine accumulation as a signal and would be repressed by HCN.\u003c/p\u003e\u003cp\u003eThe reduced levels of pyoverdine levels in all four \u003cem\u003epvdE\u003c/em\u003e mutants were not reflected in a reduced halo size in the CAS assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), which is a clear indicator that both R32 and R47 might produce and secrete one or multiple, yet unknown siderophores, in addition to pyoverdine.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe impact of the lack of pyoverdine on the ability to inhibit\u003c/b\u003e \u003cb\u003eP. infestans\u003c/b\u003e \u003cb\u003estrongly differed between the two\u003c/b\u003e \u003cb\u003ePseudomonas\u003c/b\u003e \u003cb\u003estrains\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the case of R47, the impact of pyoverdine in mycelium growth inhibition was difficult to assess, since the high inhibitory activity was maintained for all mutants, across all iron supplementation levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This was likely due to the phenazines produced by R47, which are known to be efficient biocontrol-associated molecules (Biessy \u0026amp; Filion, 2018) and have been previously demonstrated to restrict the mycelial growth of \u003cem\u003eP. infestans\u003c/em\u003e (Morrison et al., 2017). Therefore, a putative impact of pyoverdine might have been masked by the presence of phenazines in this strain.\u003c/p\u003e\u003cp\u003eEvaluating pyoverdine\u0026rsquo;s contribution to the inhibition of spore-related developmental stages of \u003cem\u003eP. infestans\u003c/em\u003e by R47 was just as difficult, this time for the opposite reason \u0026ndash; a very weak or even absent activity in the wild-type despite using higher cell densities than for R32 in these experiments (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This lack of activity differs from earlier experiments (Anand et al., 2020), but was stable across eight biological replicates and all three tested pathogen genotypes. Nevertheless, although we could not observe any significant decrease in germination percentage in R47-exposed sporangia, the length of the germ tube was significantly decreased in contact with R47 wild-type. Loss of pyoverdine did not affect this activity, in contrast to the loss of HCN, which increased it in both single and double mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eIn contrast, the results obtained \u003cem\u003ein planta\u003c/em\u003e for R47 and its mutants clearly showed an impact of both pyoverdine and HCN on disease control: while the wild-type protected the leaf discs successfully from lesion and mycelium formation, all three mutants lost their protecting activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), suggesting that both traits are important determinants of \u003cem\u003ein planta\u003c/em\u003e protection. Rather than only partially losing their protection, the single mutants were not more efficient than the double mutant, suggesting that in R47, pyoverdine and HCN could regulate each other and/or belong to a common pathway important for biocontrol activity against \u003cem\u003eP. infestans\u003c/em\u003e on leaf discs. This suggests that phenazines may not be as important to inhibit mycelium development \u003cem\u003ein planta\u003c/em\u003e as \u003cem\u003ein vitro\u003c/em\u003e, a tendency that was already observed previously (Morrison et al., 2017). Most importantly, we observed in this experiment a loss of biocontrol activity in the pyoverdine mutants, highlighting the relevance of this siderophore for the control of potato late blight. The loss of pyoverdine could have a direct impact on the iron competition, or a more indirect impact which could be linked to lower iron concentration inside the bacterial cells, which would in turn result in the dysregulation of many regulatory processes involving iron (Crosa, 1997).\u003c/p\u003e\u003cp\u003eFor R32, the results obtained present themselves quite differently, which underlines that the two strains, although both \u003cem\u003ePseudomonas\u003c/em\u003e displaying antagonistic activity against \u003cem\u003eP. infestans\u003c/em\u003e, likely employ different modes of action. In the mycelium growth inhibition under low iron conditions (no added FeCl\u003csub\u003e3\u003c/sub\u003e), the strongly reduced amount of pyoverdine had no impact on the level of inhibition in both the single and double mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This suggests that although iron competition plays a role in the interaction, which was evident from the decreased inhibition observed upon increasing iron supplementation, pyoverdine itself is not relevant for mycelial growth inhibition. More so, if pyoverdine was important for inhibiting this stage of the pathogen, we would expect a cumulative effect of the loss HCN (which resulted in reduced activity in the ∆\u003cem\u003ehcn\u003c/em\u003e mutant) and pyoverdine, but we rather measured a significantly increased activity in the double mutant compared to the ∆\u003cem\u003ehcn\u003c/em\u003e mutant, independently of iron supplementation. This increase in inhibition could be explained by the upregulation of one or multiple other biocontrol traits, such as another siderophore, as suggested above.\u003c/p\u003e\u003cp\u003eWhen looking at the plates supplemented with iron (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), we saw that R32 and its mutants secreted a reddish molecule and produced smaller and thicker colonies. The latter phenomenon of changed morphology was also observed in \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eB. cenocepacia\u003c/em\u003e (Berlutti et al., 2005), where increased biofilm formation was reported after iron addition.\u003c/p\u003e\u003cp\u003eIn zoospore release and zoospore germination, the strong decrease of pyoverdine did not reduce the activity in R32, and neither did the loss of HCN (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, the loss of both biocontrol traits did result in strong reduction or even complete loss of inhibition activity, suggesting a redundant effect of both traits.\u003c/p\u003e\u003cp\u003eWe expected results from \u003cem\u003ein planta\u003c/em\u003e assays to reflect those obtained in \u003cem\u003ein vitro\u003c/em\u003e zoospore germination assays, but it was surprisingly not the case: both single and double mutants of \u003cem\u003epvdE\u003c/em\u003e displayed conferred higher protection than R32 wild-type (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), suggesting again that in this strain, pyoverdine is not involved in the inhibition of \u003cem\u003eP. infestans\u003c/em\u003e, but that its absence would trigger the upregulation of another biocontrol trait, which remains to be identified.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBeyond iron competition \u0026ndash; a putative regulatory role of pyoverdine?\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe importance of pyoverdine as a mediator of competition for iron between \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eP. infestans\u003c/em\u003e was minor in most of our findings, except for the protective ability of R47 in leaf disc assays. This was surprising to us, since pyoverdine is known to have an especially high affinity for iron and is thought to be the main mechanism of iron acquisition in fluorescent \u003cem\u003ePseudomonas\u003c/em\u003e (Poole \u0026amp; Mckay, 2003). Since other studies have reported that pyoverdine was important for biocontrol activity (Liu et al., 2021; Ran et al., 2005; Sass et al., 2018), we deduce that pyoverdine\u0026rsquo;s importance in biocontrol activity is strongly dependent on the producer strain and the pathogen to be controlled.\u003c/p\u003e\u003cp\u003eHowever, the limited role of pyoverdine in anti-\u003cem\u003ePhytophthora\u003c/em\u003e activity of our two \u003cem\u003ePseudomonas\u003c/em\u003e strains does not mean that pyoverdine is not important for other functions in the strains. In multiple biological assays, we observed an increase in biocontrol activity when pyoverdine, or both pyoverdine and HCN were knocked out. Similarly to our previous findings that HCN is not only a toxin, but also a signaling molecule (Anand et al., 2023), there is evidence pointing to an indirect impact of the loss of pyoverdine on the R32\u0026rsquo;s overall biocontrol activity, potentially resulting from the upregulation of other biocontrol traits triggered by reduced iron concentrations in the bacteria.\u003c/p\u003e\u003cp\u003eIron is an important determinant of the whole regulatory machinery in bacteria (Hantke, 2001). Pyoverdine is not only used to acquire iron but is also believed to regulate iron homeostasis inside the cell (Schalk \u0026amp; Guillon, 2013). This implies that pyoverdine is indirectly involved in the regulatory network of \u003cem\u003ePseudomonas\u003c/em\u003e. This hypothesis is supported by the fact that pyoverdine has an important role in biofilm formation and swarming (Kang et al., 2018; Kang \u0026amp; Kirienko, 2017; Matilla et al., 2007). Matilla et al. (2007) even proposed that pyoverdine might be used in a kind of quorum-sensing mechanism in which iron would be the signal molecule and pyoverdine the regulator. In any case, the repeated observations that the lack of pyoverdine leads to higher antagonistic activity suggest that changes in iron sensing and homeostasis might be a major regulator of biocontrol activity in antagonistic \u003cem\u003ePseudomonas.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe differing activities of our two strains and their respective mutants on the different developmental stages of \u003cem\u003eP. infestans in vitro\u003c/em\u003e and \u003cem\u003ein planta\u003c/em\u003e show that biocontrol is a complex phenomenon where the roles of specific molecules can be highly dependent on the producing strain, on the target pathogen and on the conditions of the interaction (e.g. taking place in laboratory media or on plant material). Moreover, it is important to keep in mind that the results obtained on leaf discs, though \u003cem\u003ein planta\u003c/em\u003e, may not be representative of what would happen on whole plants, or even in field conditions. Understanding the true role of pyoverdine in whole plant and field assays would therefore be an important avenue to explore in future studies, as well as elucidating the mechanisms leading to increased biocontrol activity in strains deprived of pyoverdine.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn order to assess the role of pyoverdine in the antagonistic activity of two potato-associated \u003cem\u003ePseudomonas\u003c/em\u003e strains against. \u003cem\u003eP. infestans\u003c/em\u003e, we created pyoverdine mutants in different genetic backgrounds and evaluated their performance in multiple developmental stages of the oomycete. We found that the contribution of pyoverdine strongly depended on the \u003cem\u003ePseudomonas\u003c/em\u003e strain, and that its lack often resulted in higher antagonistic activity. This finding is unexpected, given pyoverdine\u0026rsquo;s high affinity for iron and given the fact that \u003cem\u003eP. infestans\u0026nbsp;\u003c/em\u003erelies on iron supply for its growth and development (Cuppett \u0026amp; Lilly, 1973). Iron is also relevant to the host plant potato, to defend itself against the oomycete upon infection (Mata et al., 2001). All this shows the importance of iron and its acquisition for the pathogenesis of \u003cem\u003eP. infestans\u0026nbsp;\u003c/em\u003eand the protective activity of biocontrol strains. This brings us to the conclusion that more studies are needed to identify the mechanisms underlying the enhanced biocontrol activity observed in pyoverdine deficient mutants and to unravel how iron homeostasis regulates biocontrol-relevant traits in plant-associated \u003cem\u003ePseudomonas.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eABC ATP-binding cassette\u003c/p\u003e\n\u003cp\u003eCAS Chrome azurol S\u003c/p\u003e\n\u003cp\u003eFUR Ferric uptake regulator\u003c/p\u003e\n\u003cp\u003eHCN hydrogen cyanide\u003c/p\u003e\n\u003cp\u003eKO Knock-out \u003c/p\u003e\n\u003cp\u003eNRPS Non-ribosomal peptide synthetase\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll referenced results can be found in results or supplementary data. Raw data can be provided upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan lang=\"EN-US\"\u003eFunding\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinancial support from the Swiss National Science Foundation (grant nr. 207917 to LW) is gratefully acknowledged.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFLH created all the \u003cem\u003epvdE\u003c/em\u003e knock-out mutants and co-supervised the work together with LW. LJ performed all experiments, analyzed the data and wrote the first draft of the manuscript. All authors contributed to the writing and editing of the manuscript. LW secured the funding.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Abhishek Anand for fruitful discussions and the sharing of his valuable expertise that helped in many aspects of the work. We also wish to thank Mout de Vrieze for her advice on the leaf discs assay, as well as S\u0026eacute;bastien Bruisson and Rudolph Rohr for their guidance regarding the statistical analyses.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAnand, A., Chinchilla, D., Tan, C., M\u0026egrave;ne-Saffran\u0026eacute;, L., L\u0026rsquo;Haridon, F., \u0026amp; Weisskopf, L. (2020). 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Siderophores are the main determinants of fluorescent Pseudomonas strains in suppression of grey mould in Eucalyptus urophylla. \u003cem\u003eActa Phytopathologica Sinica\u003c/em\u003e, \u003cem\u003e35\u003c/em\u003e(1), 6\u0026ndash;12. https://doi.org/10.1016/j.biocontrol.2004.08.007\u003c/li\u003e\n \u003cli\u003eRingel, M. T., \u0026amp; Br\u0026uuml;ser, T. (2018). The biosynthesis of pyoverdines.\u0026nbsp;\u003cem\u003eMicrobial Cell\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e(10), 424\u0026ndash;437. https://doi.org/10.15698/mic2018.10.649\u003c/li\u003e\n \u003cli\u003eSass, G., Nazik, H., \u0026amp; Penner, J. (2018). Studies of Pseudomonas aeruginosa mutants indicate pyoverdine as the central factor in inhibition of Aspergillus fumigatus biofilm. \u003cem\u003eJournal of Bacteriology\u003c/em\u003e, \u003cem\u003e200\u003c/em\u003e(1), 345\u0026ndash;362. https://doi.org/10.1128/JB.00345-17\u003c/li\u003e\n \u003cli\u003eSchalk, I. J. (2008). Metal trafficking via siderophores in Gram-negative bacteria: Specificities and characteristics of the pyoverdine pathway. \u003cem\u003eJournal of Inorganic Biochemistry\u003c/em\u003e, \u003cem\u003e102\u003c/em\u003e(5\u0026ndash;6), 1159\u0026ndash;1169. https://doi.org/10.1016/j.jinorgbio.2007.11.017\u003c/li\u003e\n \u003cli\u003eSchalk, I. J., \u0026amp; Guillon, L. (2013). Pyoverdine biosynthesis and secretion in Pseudomonas aeruginosa: Implications for metal homeostasis. \u003cem\u003eEnvironmental Microbiology\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e(6), 1661\u0026ndash;1673. https://doi.org/10.1111/1462-2920.12013\u003c/li\u003e\n \u003cli\u003eSchneider, C. A., Rasband, W. S., \u0026amp; Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis.\u0026nbsp;\u003cem\u003eNature Methods\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(7), 671\u0026ndash;675. https://doi.org/10.1038/nmeth.2089\u003c/li\u003e\n \u003cli\u003eSi-Ammour, A., Mauch-Mani, B., \u0026amp; Mauch, F. (2003). Quantification of induced resistance against Phytophthora species expressing GFP as a vital marker: \u0026beta;-aminobutyric acid but not BTH protects potato and Arabidopsis from infection. \u003cem\u003eMolecular Plant Pathology\u003c/em\u003e, \u003cem\u003e4\u003c/em\u003e(4), 237\u0026ndash;248. https://doi.org/10.1046/j.1364-3703.2003.00168.x\u003c/li\u003e\n \u003cli\u003eVisca, P., Imperi, F., \u0026amp; Lamont, I. L. (2007). Pyoverdine siderophores: from biogenesis to biosignificance. \u003cem\u003eTrends in Microbiology\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e(1), 22\u0026ndash;30. https://doi.org/10.1016/j.tim.2006.11.004\u003c/li\u003e\n \u003cli\u003eWang, W., \u0026amp; Long, Y. (2023). A review of biocontrol agents in controlling late blight of potatoes and tomatoes caused by Phytophthora infestans and the underlying mechanisms. \u003cem\u003ePest Management Science\u003c/em\u003e, \u003cem\u003e79\u003c/em\u003e(12), 4715\u0026ndash;4725. https://doi.org/10.1002/ps.7706\u003c/li\u003e\n \u003cli\u003eYeterian, E., Martin, L. W., Guillon, L., Journet, L., Lamont, I. L., \u0026amp; Schalk, I. J. (2010). Synthesis of the siderophore pyoverdine in Pseudomonas aeruginosa involves a periplasmic maturation. \u003cem\u003eAmino Acids\u003c/em\u003e, \u003cem\u003e38\u003c/em\u003e(5), 1447\u0026ndash;1459. https://doi.org/10.1007/s00726-009-0358-0\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pseudomonas, Phytophthora infestans, hydrogen cyanide, pyoverdine, siderophores, biocontrol, potato","lastPublishedDoi":"10.21203/rs.3.rs-7139568/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7139568/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe oomycete \u003cem\u003ePhytophthora infestans\u003c/em\u003e has been causing detrimental yield losses over the last 200 years and to this day, controlling measures heavily rely on synthetic pesticides. In view of their environmental toxicity, biological control represents an attractive alternative to fight this pathogen. \u003cem\u003ePseudomonas\u003c/em\u003e strains are known to produce a large arsenal of secondary metabolites conferring protection against several crop diseases. Next to biocontrol traits causing direct pathogen inhibition, such as antibiotics and toxins, siderophores are considered important mediators of competitive inhibition of plant pathogens by \u003cem\u003ePseudomonas\u003c/em\u003e. However, whether siderophore production plays any role in the biocontrol of the late blight causing agent \u003cem\u003eP. infestans\u003c/em\u003e has not yet been investigated. In this study, we focused on two \u003cem\u003ePseudomonas\u003c/em\u003e strains, R32 and R47, which have been previously characterized as successful antagonists against \u003cem\u003eP. infestans\u003c/em\u003e. Both strains produce pyoverdine, thus the aim of this study was to evaluate the role of pyoverdine in the inhibition of \u003cem\u003eP. infestans\u003c/em\u003e in both strains. For this purpose, we created pyoverdine-deficient mutants by knocking-out \u003cem\u003epvdE\u003c/em\u003e, the periplasmic ferribactin exporter. We did this in both wild-type and HCN-deletion backgrounds for the two strains. These mutants were then tested for loss of antagonistic activity against \u003cem\u003eP. infestans\u003c/em\u003e in several \u003cem\u003ein vitro\u003c/em\u003e assays targeting different developmental stages of the pathogen life cycle, as well as in leaf disc assays to assess the relevance of pyoverdine \u003cem\u003ein planta.\u003c/em\u003e Our results indicate that pyoverdine plays a different role in both \u003cem\u003ePseudomonas\u003c/em\u003e strains: in leaf disc assays, lack of pyoverdine completely suppressed the ability of R47 to restrict symptom development, but it increased the protective efficacy of R32. In this latter strain, the lack of pyoverdine alone did not diminish its ability to inhibit the pathogen\u0026lsquo;s mycelium or spores, but when combined to the loss of HCN, it either led to a complete loss of inhibition in spore assays, or to stronger inhibition in mycelium assays. These results suggest an interplay between HCN and pyoverdine and the upregulation of a yet unknown mechanism underlying the higher \u003cem\u003ein planta\u003c/em\u003e protective efficacy observed in R32 pyoverdine-deficient mutants.\u003c/p\u003e","manuscriptTitle":"Evaluating the contribution of pyoverdine to the anti-Phytophthora activity of two potato-associated Pseudomonas strains","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-07 11:20:38","doi":"10.21203/rs.3.rs-7139568/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7f1b7eeb-140e-4aa5-881e-b960efbf59f5","owner":[],"postedDate":"August 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52678835,"name":"Biological sciences/Microbiology"},{"id":52678836,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2025-08-29T06:09:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-07 11:20:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7139568","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7139568","identity":"rs-7139568","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}