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Chemicals were purchased from Sigma–Aldrich (Switzerland) with the exception of 1-dodecene (Dr. Ehrenstorfer GmbH, Germany) and 2-acetylfuran (Alfa Aesar, Germany). Luria-Bertani (LB) medium was prepared by dissolving 20 gl^-1 of Difco LB Broth, Lennox (BD) and adding 15 gl^-1 agar (Agar Agar, ERNE surface AG). Rye agar (RA) was prepared by gently boiling 200 g rye grains in 1.5 l tap water for 1 h. The liquid was then filtered through a sieve (1.5 mm mesh) and filled up to the end volume of 1 l with tap water and supplemented with 5 gl^-1 D-glucose. 20 gl^-1 agar were added. Petri dishes were filled using a plate-pouring machine (Mediajet, Integra Biosciences) with 18 ml of medium in standard Petri dishes (94 mm × 16 mm, Greiner Bio-One).

A P. infestans polypore isolate sampled in 2001 (provided by H. Krebs, Agroscope) was used for all experiments. This isolate had been maintained as mycelial culture on RA and regularly transferred to potato slices for host passage. Petri dishes were sealed with Parafilm M (BEMIS Flexible Packaging) and incubated or stored in the dark at 18°C.

Most bacteria were isolated and maintained as in (Hunziker et al., 2015). Pseudomonas protegens CHA0 and its corresponding hcn^A- mutant CHA77 were obtained from Prof. Dr. Dieter Haas (University of Lausanne). Bacterial strains were routinely grown on LB and kept at ^-80°C in 25% glycerol for long-term storage.

In order to elucidate the phylogenetic relationships of the 16 candidate Pseudomonas sp. strains with respect to additional selected Pseudomonas reference strains, the sequences of four major housekeeping genes including 16s rRNA, gyrB, rpoD, and rpoB (Mulet et al., 2010; Gomila et al., 2015) were extracted from an Illumina MiSeq paired end (2x 300 bp) sequencing effort aimed at describing the gene inventory of these strains. The contigs that resulted from de novo genome assembly using the Spades algorithm (Bankevich et al., 2012) were then annotated with the software Prokka (Seemann, 2014), prior to retrieving the sequences of the four housekeeping genes and subsequently concatenating them in the order 16s rRNA, gyrB, rpoD, and rpoB. The MEGA (Molecular Evolutionary Genetic Analysis) software (Kumar et al., 2008) was used to construct a maximum-likelihood-based phylogenetic tree from the concatenated sequences of the four genes of 16 strains, as well as of nine additional reference Pseudomonas strains, including P. protegens CHA0. All generated sequences were deposited to GeneBank under the accession numbers KT890284 - KT890343¹.

The volatiles of sixteen selected strains were collected and analyzed by gas chromatography-mass spectrometry (GC/MS) using closed-loop-stripping analysis (CLSA) as described earlier (Hunziker et al., 2015) using a modified apparatus design (see Supplementary Figure S5). The strains were pre-grown at 18°C on LB-agar plates for 4 days before single colonies were resuspended and adjusted to a density of OD_570nm = 1 in sterile water. Bacterial samples were cultured by inoculating 50 μl of cell suspensions, spread as a layer using sterile glass beads into 4 cm glass Petri dishes to avoid plastic volatile contaminants and grown for 24 h at 25°C in the CLSA apparatus under constant air flow. Uninoculated LB-agar glass plates were used as controls. Trapped volatiles were extracted from the charcoal filter by rinsing the filter three times with 20 μl dichloromethane (≥99.8%, Merck, Germany). The headspace extracts were subsequently adjusted to 100 μl with dichloromethane and analyzed by GC/MS. Each experiment was repeated three times. Analyses by GC/MS were performed on a Varian CP3800 gas chromatograph (Varian, Walnut Creek, CA, USA) connected to a triple quadrupole mass spectrometer (Varian 1200, Varian). Separation by GC took place on a Rtx-5Sil MS capillary column (30 m, 0.25 mm i.d., 0.25 μm film thickness) from Restek (Bellafonte, PA, USA). As a retention gap, a 2 m Siltek guard column (0.53 mm i.d., Restek) was mounted in front of the separation column. Helium was used as a carrier gas at a constant flow of 1 ml.min^-1. The samples were injected on-column into a programmable temperature vaporization injector (temperature program: 50°C for 0.1 min, to 300°C at 200°C.min^-1, 30 min at 300°C, to 160°C at 20°C.min^-1, 20 min at 160°C). The oven temperature was programmed as follows: 10 min at 40°C, to 320°C at 25°C.min^-1, 1 min at 320°C. The transfer line temperature was set at 200°C. Analyte detection by MS was conducted in the electron impact mode with 70 eV ionization energy, at a source temperature of 250°C. Full scan monitoring (scan time 0.7 s) was performed in the m/z mass range from 35 to 350. Compounds were identified by comparison of mass spectra to database spectra (NIST 08 and pure commercial reference compounds), and comparison of the retention times and mass spectra previously obtained (Hunziker et al., 2015).

The effect of selected pure compounds of the VOCs blends on mycelial growth, sporangia production and germination, zoopore release, motility and germination of P. infestans was assessed as follows: 5 mm agar plugs from the edge of actively growing mycelial colonies were placed downward-faced in the center of fresh RA plates. Definite quantities of the test compounds or dilutions in dimethylsulfoxide were applied on PTFE/silicone septa (8 mm, Supelco) and placed in the center of the Petri dish lid so the test compounds faced the mycelial plug. Plates were sealed with Parafilm M and incubated upside-down in the dark at 18°C. Mycelial growth was monitored 7 days after inoculation by taking photographs and total mycelial area was further assessed using ImageJ. At the end of a 9-day incubation period, the plates were opened and sporangia were collected using 5 ml room temperature sterile water by gently rubbing the mycelial mat with a sterile glass rod. A 500 μl aliquot of the resulting suspension was dispensed into a 24-well polycarbonate plate. The number of produced sporangia and rate of germination was assessed under the binocular right after collection and 24 h later, respectively. The same procedure was used to produce zoospores, except that 10 ml of ice-cold water was laid on top of the mycelial mat and kept at 4°C for 2 h. After this incubation period, the suspension was left at room temperature for 20 min prior to collection in order to allow zoospore release to occur. A 500 μl aliquot of the resulting suspension was dispensed into a 24-well polycarbonate plate. The quantity of produced zoospores and zoospore motility was assessed under the binocular right after collection. Zoospore encystment and subsequent germination was assessed 24 h later.

To evaluate the effect of pure compounds on direct sporangial germination, sporangia were collected in 10 ml room temperature sterile water from 9 days-old mycelial plates and adjusted to a concentration of 200’000 sporangia.ml^-1 in Eppendorf tubes supplemented with the adequate chemical treatment. No vortexing was applied in order to avoid triggering germination. After 1 day of incubation at 20°C in the dark, series of 10 μl droplets of the suspension were mounted on glass slides and photographed under the microscope. The number of closed, open and germinating sporangia were counted using ImageJ. In both experimental setups, the inhibitory concentration yielding 50% inhibition (IC₅₀) of mycelial growth or sporangial germination for each treatment was calculated through extrapolation from the curve-fitted plots.

Zoospores from preparations obtained as described above were used to follow the trajectories of individual cells over time. Image sequences were taken for 10 s, using a Wild Heerbrugg MDG17 binocular (Wild Heerbrugg, Switzerland) coupled to a Leica DFC290 Camera using the LAS software v4.6.2 (Leica, Germany). Images were next filter-transformed using ImageJ to achieve high contrast. Single particle tracking over the segmented images was performed using the ImageJ TrackMate plugin², (Jaqaman et al., 2008) using semi-automated default parameters. Chemical treatments were performed immediately prior to observations and image capture to avoid any bias from the decrease of zoospore motility occurring in control conditions.

Phytopthora infestans 208 m² was obtained from Prof. Dr. F. Mauch (University of Fribourg) and maintained on RA plates as described above. Sporangial yield was increased by exposing the Petri dish to natural light for 8 h at days 7 and 8 after inoculation. Sporangia were collected as described above 9 days after inoculation and GFP fluorescence was visualized and digital micrographs acquired using a Zeiss Axiovert 200 M stereomicroscope equipped with adequate fluorescent source and filters. The GFP signal mean gray intensities of individual sporangia were extracted using a custom-made segmentation macroinstruction protocol to estimate the signal intensity distribution at the population level.

A 10 μL drop of a water suspension of 125’000 sporangia.mL^-1 was applied in the middle of the abaxial side of a ø17 mm leaf disk cut from 1 month old potato plants cv. Victoria. Leaf disks were placed on a soaked filter paper in standard Petri dishes. Definite amounts of the test compounds were applied on PTFE/silicone septa (8 mm, Supelco) and placed in the center of the Petri dish, 3 cm away from each leaf disk. Petri dishes were closed with Parafilm M, placed in a lightproof plastic box and incubated at 18°C for a period of 8 days. P. infestans infection was then visualized and acquired under the binocular.

Data were analyzed using the GraphPad Quikcalcs tools³, GraphPad Prism 5 software and Microsoft Excel software.

In order to correlate the production of specific bVOCs to the inhibitory potential of the 16 different Pseudomonas strains we previously screened, showing various VOCs-mediated inhibition against P. infestans (Hunziker et al., 2015), we trapped and identified the different chemical species they emitted. With the underlying hypotheses that the most active strains should produce either (1) a different set of compounds or (2) differential amounts of specific compounds, we cultured and further analyzed the bacterial emissions in standardized conditions. All tested strains were inoculated with the same density of cell suspension and let grow for the same amount of time under identical culture conditions. We chose to collect the bVOCs over a 24 h incubation period (i.e., until late stationary phase) with the aim to maximize the chance of recovering chemoprofiles that would best represent the P. infestans growth-inhibiting bVOC blends occurring in our previously published dual-assays (Hunziker et al., 2015). Furthermore, a steady closed-loop-stripping apparatus allowed us to trap and extract the emitted chemical species with high reproducibility. Consequently, although the GC/MS spectra obtained do not report quantitative amounts of individual chemical species, they capture very comparable relative profiles of the different test strains’ biogenic emissions (Figure 1 and Supplementary Figure S1).

Under these conditions, we were able to retrieve a large majority of the volatile compounds identified earlier and obtained similar chromatograms (Hunziker et al., 2015). However, slight changes in the gas chromatograph setup and parameters allowed us to get a better insight into the smallest and more hydrophilic components of the volatile emissions from plant-associated Pseudomonas. To our surprise, we discovered that in many of the tested strains the sulfur-containing compound dimethyldisulfide (DMDS) was produced in similar or higher amounts as 1-undecene (Figure 1), which we have described earlier as being the major chemical in the bacterial VOCs mixture (Hunziker et al., 2015). When GC peak area of definite amounts of pure DMDS and 1-undecene where compared to test spectra, a 24 h collection period yielded micrograms of both compounds, thus underlining the capacity of bacterial strains to accumulate high concentrations of VOCs in the headspace. In the vast majority of strains, 1-undecene, DMDS, 4-hydroxy-2-pentanone and benzaldehyde were the major peaks of the emission spectra. No particular chemical patterns could be unequivocally found between the different strains included in this study, neither by clustering the spectral data according to the strains’ phylogeny (Figure 1, and Supplementary Figure S2) or origin of isolation (rhizosphere vs. phyllosphere, Supplementary Figure S1). We also did not observe a straight correlation between the presence or the amounts of a given chemical species and the full VOCs blends effects of the strains on P. infestans radial growth (Hunziker et al., 2015, Figure 1). While the strongest P. infestans growth inhibition was as expected caused by HCN-producing strains (Hunziker et al., 2015), the individual volatile compounds that could contribute to the P. infestans inhibition triggered by non-HCN producing strains were not obvious to deduce. However, comparison of the emission spectra of the biocontrol P. fluorescens CHA0 (Voisard et al., 1989) with those of its corresponding HCN deficient, hcnA^- mutant CHA77 (Laville et al., 1998) revealed differences in the metabolite production (Figure 2). Indeed CHA77 produced lower amounts of DMTS and 1-undecene, and significantly higher amounts of DMTS and MMTS. In addition, we observed a set of two or more yet uncharacterized compounds (rt = 26.8 min) that seem to be common products from non-cyanogenic bacteria (Figure 1). Likewise, the CHA0 strain produced higher amounts of limonene and two supplementary compounds that await formal identification. From the same initial inoculum density, several strains such as R32, R84, CHA0, S04, or S19 had apparent higher levels of volatile production that could be explained by a higher metabolism or a better adaptation to the culture conditions (Figure 1). Nevertheless, the phylogenetic group comprising R74, R95, S04, S19, and S34 seemed to have repeatedly produced smaller amounts of 4-methyl-2-pentanone and 4-hydroxy-4-methyl-2-pentanone. Moreover, strains that produced the lowest, or not detectable amounts of DMDS, R01, R32, R47, S22, and S34, showed also the lowest detected amounts of DMTS and MMTS. Finally, the strain R75, that displayed a clearly dissimilar chemoprofile, was formally identified as belonging to the Flavobacterium genera, and not as previously reported to Pseudomonas (Hunziker et al., 2015), supporting the concept that bacteria could be identified at the species level via their volatile chemical signature (Thorn et al., 2011).

The high number of sampled Pseudomonas volatile profiles and their high similarity prompted us to investigate the contribution of a subset of individual chemical species to the previously observed growth inhibition of P. infestans by VOCs inhibition. Although not exhaustive, the list of tested, commercially available compounds we present here covers a large portion of the recorded Pseudomonas spectra (Figure 1 and Table 1). While our pharmacological screen revealed that half of the tested compounds showed mild to low growth inhibition of the plant pathogen at high applied amounts, only few compounds possessed strong inhibitory activity (i.e., an IC₅₀ below 1 mg, reaching at least 30% inhibition and a R² > 0.6) against P. infestans mycelial growth. Nitropentane, isovaleric acid, undecanal, phenylpropanedione, propiophenone, dimethyl trisulfide (DMTS) and S-methyl methanethiosulfonate (MMTS) showed total inhibition of P. infestans growth at 1 mg. Although 1-undecene did not perform very well in hindering P. infestans growth within the applied amounts, its inhibitory activity was found to be close to those reported earlier (Hunziker et al., 2015). In our hands, the bacterial quorum-sensing-related molecules 2’-aminoacetophenone and acetophenone had low influence on growth of the pathogen, reaching merely about 30% mycelium area reduction. In contrast, cyclic ketones sharing a similar backbone such as propiophenone, and phenylpropanedione were amongst the most potent chemicals assayed in this study, with IC50 values ranging from 10 μg to 0.5 mg. Strikingly, nearly all sulfur-containing VOCs showed a great mycelial growth inhibition potential. From all assessed chemical species, MMTS was the most potent with an estimated IC₅₀ in the low nanogram range. We did not observe specific apparent phenotypic defects in P. infestans upon exposure to the tested pure chemicals, with the exception of 2-undecanone (and to a lesser extent 2-decanone) treatments, which resulted in a dose-dependent densification of the mycelial mat and extended mycelium aerial growth, resulting in a “fluffy” appearance of the colony (Supplementary Figure S3).

Phytophthora infestans is a complex and adaptive eukaryotic lifeform that can spread and survive in multiple diverse forms through its life cycle, inside and outside its Solanaceae hosts (Fry, 2008; Fry et al., 2015). While mycelium growing on nutrient rich media appeared robust to the chemical treatments, we examined the impact of the individual volatiles exposure on both asexual forms that serve as infection vectors, namely sporangia and zoospores (Fry, 2008).

We then assessed the ability of our P. infestans isolate to produce sporangia and discharge zoospores after the end of the mycelial growth period in presence of the pure volatiles. We also monitored the germination rate of both spore types and the motility of zoospores. Obviously chemicals that greatly impaired Phytophthora growth, nitropentane, isovaleric acid, undecanal, DMTS, MMTS, propiophenone and phenylpropanedione had a strong impact on sporangia production, and consequently on zoospore numbers (Figure 3). However, other compounds that did not greatly impede mycelial growth, such as the simple ketones 3-hexanone, 2- and 4-heptanone, 2-decanone and 2-undecanone, 4-hydroxy-methyl-2-pentanone as well as the alkenes 1-undecene and 1-dodecene significantly impacted sporangia production (Figure 3), suggesting that these mid-to-long hydrocarbon chains could specifically interfere with this process. At the highest amounts tested, diphenylamine and 2-acetylthiazole also decreased sporangial formation. The same set of chemicals also decreased the germination rate of sporangia, though this measurement does not depend on sporangia production, indicating that the spores may have been hindered during their developmental process or that non negligible amounts of the supplied VOCs were extracted during sample preparation. Based on the data displayed in Figure 3, limonene, 1-dodecene, and S-methylbutanethioate seem to have a specific effect on sporangia germination after mycelium exposure. Logically, the production of zoospores largely followed the pattern of sporangia production in terms of inhibition mediated by individual VOCs. None of the tested compounds seemed to specifically block this process. Further, zoospores released from sporangia freely swam in most treatments and exhibited no particular defects, with the notable exception of treatments of the mycelium with high amounts of MMTS, where a large number of zoospores underwent cell lysis. Nevertheless, exposure to 2-undecanone, limonene, 1-undecene, diphenylamine, S-methylbutanethioate, acetophenone, or phenylacetone during mycelial growth significantly decreased zoospore motility. Nitropentane, isovaleric acid, undecanal, as well as propiophenone and phenylpropanedione had also a strong negative impact on swimming in the highest treatments, while DMDS, DMTS, and MMTS showed the greatest inhibition. As for sporangia, the influence of pure compounds on the formation of germ tubes from encysted zoospores mostly resembled the effect on zoospore production. Taken together, these results suggest that Pseudomonas bVOCs have the potential to alter and impede P. infestans development, and that bioactive compounds mostly act similarly on the different lifeforms of the pathogen (mycelium, sporangia, zoospores) rather than specifically interfering with only one or the other.

The fungicides currently in use to constrain late blight spreading in potato crops vary in their activity against the different developmental stages in the life cycle of Phytophthora species, acting either against mycelial growth, zoospore release, zoospore motility, or germination of spores (Gisi and Cohen, 1996; Cohen and Gisi, 2007). Therefore, in order to select for P. infestans biocontrol biogenic volatiles, we focused this work on direct sporangia germination for the following reasons: (1) the P. infestans population shift observed since the 1980’s displays A2 mating type-dominated populations that tend to favor direct germination (Li et al., 2013; Fry et al., 2015), (2) sporangia are easily generated and collected in high yields, thus allowing larger screening efforts in the laboratory; (3) sporangia germination is easy to score and requires minimal equipment. We collected mature sporangia from control-grown P. infestans colonies and incubated them in serial dilutions of the test chemicals. After 24 h of treatment, about half of the chemicals tested showed inhibition of the germination process at 1 mg treatments (Table 2). However, the inhibitory effect rapidly dropped in most cases at lower drug amounts. Volatile species that demonstrated a significant inhibition potential (i.e., IC₅₀ < 1 mg, reaching at least 30% inhibition and a R² > 0.6) were 3-hexanone, 1-dodecene, isovaleric acid, S-methylbutanethioate, MMTS, furfuryl alcohol, acetophenone, phenylpropanedione, and 2-acetylthiazole (Table 2). Although 1-undecene and nitropentane treatments extrapolations did not meet these criteria, both molecules displayed satisfactory inhibitory power. Amongst these compounds, nitropentane, isovaleric acid and MMTS, as well as diphenylamine, were the only species to completely prevent the initiation of sporangia germination at 1 mg treatments. All other compounds displayed various degrees of germ tube elongation. Interestingly, the ketones 3-hexanone, 2-undecanone and, to a lesser extent, 2-decanone often led to decreased germ tube lengths and swellings or bursts of the growing germ tube tip, as well as ectopic initiation site (Supplementary Figure S4). This suggests that accumulation of these molecules specifically hinders the normal growth of the germ tube but not its initiation per se. We conclude that the sporangial germination process is widely sensitive to biogenic volatiles. Although the amounts employed in these experiments do probably not reflect the bVOCs quantities produced in the rhizosphere or phyllosphere in addition to the poor water solubility of some test chemicals, it seems reasonable to postulate that several individual volatiles contribute synergistically to the activity of the whole blend of bVOCs.

Out of the forty single volatiles we tested, two compound groups retained our attention for their potent differential activity on P. infestans (Figure 3 and Table 2): the sulfur-containing compounds DMDS, DMTS and MMTS and the simple ketones 3-hexanone, 2-decanone, and 2-undecanone. The first compounds seem able to block P. infestans growth and development while the latter specifically hinders sporangial germination when directly applied to the sporangia. We therefore investigated further the mode of action of DMTS, MMTS, and 3-hexanone using P. infestans 208 m², a strain constitutively expressing the GFP fluorescent protein (Si-Ammour et al., 2003). With the aim to examine if the tested VOCs show sporicidal activities, we compared the GFP signal intensities of sporangial populations exposed to increasing amounts of volatiles for 24 h (Figure 4A). While control sporangia populations displayed a broad distribution of mean fluorescence signal intensities, ranging from close to background to highly fluorescent sporangia, both DMTS and MMTS treatments shifted the population fluorescence to low signals, presumably indicating that sporangia died during treatments. This hypothesis was further verified by treating ungerminated and germinated, GFP-expressing Phytophthora sporangia and cystospores with increasing amounts of these sulfur compounds. After 20 min of exposure with 1 mg MMTS, the GFP signal dramatically decreased in both spores and germ tubes (Figure 4B) and appeared practically abolished after 40 min without affecting the cellular structure while control treatments showed constant GFP signals over the same time frame. This suggests that MMTS directly blocked the spores’ cellular activity. In addition, propidium iodide cell viability tests confirmed the death of treated cells (results not shown). Since cell wall-free zoospores are recognized as being highly sensitive to exogenous chemicals (Judelson and Blanco, 2005; Chen et al., 2012) we tested the zoosporicidal potential of MMTS. We subjected fresh, freely swimming zoospore preparations to serial dilutions of MMTS and assessed their motility under the binocular. Representative zoospore trajectories obtained via computer-assisted single particle tracking are shown in Figure 4C. Surprisingly, MMTS treatments displayed a significant decrease of zoospore swimming at concentrations as low as 1 fg.ml^-1, thus revealing a strong potency (data not shown). At highest concentrations (>1 μg.ml^-1) MMTS immediately blocked zoospore motility followed by cell lysis. Moreover, during the course of these experiments carried out in 24-well plates, the volatile diffusion of MMTS from the high concentration wells was sufficient to inhibit completely zoospore motility in distant control wells.

Conversely, exposure of GFP-expressing sporangia to 3-hexanone led to a population shift toward sporangia with higher fluorescent signals that did not correlate with sporicidal effects (Figure 4A). At highest amounts (>10 μg.ml^-1), 3-hexanone treatments triggered a reorganization of the inner sporangial GFP signal comparable to that of germinating sporangia although without germ tube production (Figure 4D). These observations, together with the abnormalities observed during sporangial germination described above (Supplementary Figure S4) seem to indicate that 3-hexanone is not sporicidal but rather directly interferes with the germination process of sporangia.

The ultimate goal of this work was to investigate whether biogenic volatiles produced by native potato-associated bacteria could be exploited to control late blight infection in planta. Consequently, we used our standardized laboratory leaf disk assay to assess the impact of pure VOCs on disease progression. Potato leaf disks inoculated with a sporangial suspension droplet were incubated for 8 days in presence or absence of 1 mg of DMDS, DMTS, or MMTS (Figure 5). At the end of the experiment, inoculated unexposed controls displayed strong symptoms with a dense mat of sporangiophores while treatments with the sulfur compounds reflected their respective inhibitory potential deduced from the experiments described above. At this amount, exposure to DMDS drastically reduced sporangiophore production and both DMTS and MMTS treatments prevented disease development. Regarding the facts that our leaf disk experimental design favors the direct sporangial germination in P. infestans infection, this suggested that both DMTS and MMTS treatments blocked this event. This assumption was further confirmed by microscopic observations (data not shown).