Forest tree extracts induce resistance to Pseudomonas syringae pv. tomato in Arabidopsis | Scientific Reports

Scientific Reports volume 14, Article number: 24726 (2024) Cite this article

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The widespread use of conventional pesticides for plant pathogen control poses significant risks to human health and the environment, and it is therefore crucial to develop environmentally friendly, human-safe alternatives to these products that offer a sustainable approach for crop protection. Here, we examined the potential of ethanolic extracts from four forest tree species for their antibacterial activity against the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) and their ability to trigger effective defense responses in the model plant Arabidopsis thaliana. The extracts exhibited direct toxic effects against Pst and triggered the expression of defense-related genes naturally induced by oxidative stress cues or the defense elicitor salicylic acid in leaf tissue. The direct antibacterial effects of the tree extracts, together with their defense gene-inducing effects in planta, resulted in a strong host plant-protecting effect against Pst. These findings suggest the eventual effectiveness of forest tree extracts as plant protectants against the bacterial pathogen Pst. They also suggest the potential of these extracts as a sustainable, eco-friendly alternative to conventional pesticides for the management of economically important plant pathogens.

Plant diseases pose a significant threat to agricultural and horticultural crops worldwide, resulting in substantial losses in crop productivity, quality and economic value1. The use of conventional pesticides, such as fungicides and bactericides, is widespread for controlling plant pathogenic fungi and bacteria2,3. However, their extensive use has faced increasing criticism due to the various problems they cause to agriculture, human health and the environment4,5,6. It is therefore crucial to urgently develop environmentally friendly, human-safe alternatives to conventional pesticides that offer a sustainable approach to plant pathogen control.

Over the past two decades, several studies have demonstrated the potential of plant extracts in limiting the development of plant diseases and highlighted their potential as a sustainable alternative to conventional pesticides7,8,9,10,11. Numerous studies have reported the efficacy of such extracts to prevent microbial diseases on cultivated plants, caused notably by oomycete and fungal pathogens such as Phytophthora infestans, Oidium spp., Puccinia triticina, Fusarium oxysporum and Botrytis cinerea, or by bacterial pathogens such as Pseudomonas cichorii, Xanthomonas campestris and Clavibacter michiganensis12,13,14,15,16,17. Disease control using plant extracts has been explained in several cases by their direct toxic effects on the pathogens12,14,17,18. It has also been associated with the induction of plant natural defenses leading to the accumulation of antimicrobial proteins and organic toxic compounds in host plant tissues12,13,15,19.

Here, we investigated the plant protective potential of ethanolic extracts from twigs or leaves of forest tree species, namely eastern hemlock (Tsuga canadensis), eastern red cedar (Juniperus virginiana), English oak (Quercus robur) and red pine (Pinus resinosa). Studies were conducted both to monitor the direct antibacterial activity of the extracts, and to assess their ability to activate host plant natural defenses eventually detrimental to plant pathogens. We used as a pathosystem the model plant Arabidopsis thaliana infected with Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000)20,21, the causal agent of bacterial speck in tomato cultivation setups worldwide22.

Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were first determined by standard procedures16,18 to evaluate the direct toxic effects of the tree extracts against Pst DC3000 (Table 1). MIC values, that refer to the lowest concentrations of extracts at which no metabolic activity of Pst DC3000 was observed following a 24-h incubation period, ranged from 0.8 to 25 mg mL−1 from one extract to another, with English oak extract showing the lowest value (at 0.8 mg mL−1) and hence the strongest antibacterial effect. MBC values, that refer to the lowest concentrations of extracts killing ≥ 99.9% of the bacteria following a 24-h incubation period, ranged from 25 to more than 50 mg mL−1. Again, the English oak extract was the most potent, with an MBC of 25 mg mL−1 compared to MBC values greater than 50 mg mL−1 for the other three extracts. As expected, given the negligible direct toxic effects of this compound23, the salicylic acid (SA) functional analogue benzothiadiazole (BTH) (commercialized as ACTIGARD™ 50WG) used as a positive control for SA-inducible defense gene inductions (see below) showed no measurable toxic effect against the bacterium, with MIC and MBC values both greater than 50 mg mL−1.

Previous studies have reported the pharmacological and therapeutic benefits of forest tree extracts in animals and humans, notably including extracts of English oak and related species of the Quercus genus used at working concentrations of 10 to 1,000 mg mL−1 24,25,26. In complement, our data suggest the potential of such extracts as antibacterial phytosanitary agents in crop protection, similar to Da Silva et al27 reporting the antibacterial activity of extracts from twelve Brazilian medicinal plants against the plant pathogens Acidovorax citrulli, Pectobacterium carotovorum subsp. carotovorum, Ralstonia solanacearum and X. campestris pv. campestris. Several secondary compounds in plants exhibit antimicrobial activity, such as for instance various terpenes, alkaloids and flavonoids found in red pine, English oak and other forest trees28. Studies will be welcome in coming years to identify the antibacterial determinants of the tree extracts considered in this study, notably for the English oak extract showing relatively low MIC and MBC values.

Defense gene-inducing effects of the tree extracts were assessed using pathogenesis reporter line PR1::GUS29, a transgenic Arabidopsis line engineered to express reporter protein ß-glucuronidase (GUS) under the control of the PR1 promoter, an SA-inducible promoter driving the expression of pathogenesis-related protein 1 (PR1) upon biotroph or hemibiotroph pathogen infection (Fig. 1). GUS expression in the PR1::GUS line was induced by all extract treatments, albeit at different levels depending on the extract. GUS staining was observed in all organs, including the cotyledons, the hypocotyl and the true leaves (Fig. 1A). Eastern hemlock, eastern red cedar and red pine extracts exhibited strong PR1 promoter-inducing effects compared to the negative control treatment, roughly comparable to the inducing effect of SA functional analogue BTH used as a positive control for SA-inducible PR1 gene expression30. By comparison, plants treated with the English oak extract showed a faint GUS signal, indicating a weaker promoter-inducing effect compared with the other three extracts (Fig. 1A, B).

PR1 gene induction in Arabidopsis PR1::GUS line treated with forest tree extracts. (a) Representative seedling images showing GUS staining activity following extract treatments. (b) GUS staining intensity per plant as determined by image densitometry of the treated plants. Plants were treated with the tree extracts at working concentrations of 12.5 and 25 mg mL−1, with BTH (ACTIGARD™ 50WG) at 0.5 mg mL−1 (positive control), or with sterile water used as negative control (CTRL). Data on panel b are expressed relative to the control (relative value of 1.0). Each value is the mean of three replicates (with six plants per replicate) ± se. Means with the same letter are not significantly different (post-ANOVA LSD, with an alpha threshold value of 5%). EH, eastern hemlock; ERC, eastern red cedar; EO, English oak; RP, red pine.

RT-qPCR assays were carried out for PR1 and additional SA-inducible genes (Table 2) to confirm the differential inducing effects of tree extracts on endogenous PR1 gene promoter activity (see Fig. 1) and to highlight a possible induction of the SA defense pathway eventually triggering host plant resistance to pathogen attack31,32,33,34 (Table 3). DNA primers were also used for NPR1, the DNA sequence of master regulator nonexpressor of pathogenesis-related genes 1 (NPR1) protein35,36. As expected, given the reported limited effect of SA on NPR1 expression in Arabidopsis37,38, BTH and the four tree extracts had no significant effect on the abundance of NPR1 transcripts. By comparison, the extracts strongly induced the transcription of endogenous PR1 in treated plants relative to the control, similar to BTH and in accordance with their inducing effects on GUS expression in the PR1::GUS reporter line (Fig. 1). The WRKY70 gene, coding for SA signaling-inducing transcription factor WRKY7039, was also induced at various rates by BTH and the tree extracts, similar to SA-inducible genes for antimicrobial proteins glucan endo-1,3-beta-d-glucosidase (PR2)40 and Kunitz trypsin inhibitor 4 (KTI4, also referred to as KTI1)41 produced downstream along the SA defense pathway. For instance, transcript numbers were increased by up to 7.8-fold for WRKY70 in BTH-treated plants, or by 7.4-fold for KTI4 and 3.4-fold for PR2 in plants treated with the eastern red cedar and eastern hemlock extracts, respectively.

In line with the differential effects of plant extracts on GUS expression, the gene inducing effects were variable from one extract to another, from a null, even negative effect for some extracts used at low concentration to more than 100% increases in more than 20 extract dose–gene combinations out of 40 combinations assessed (Table 3). Also in line with the GUS expression assay, endogenous PR1 transcriptional induction by the English oak and red pine extracts, with transcript levels 0.8- to 2.2-fold compared to the control treatment, were weaker than the 3.0- to 5.1-fold inductions observed for the eastern hemlock and eastern red cedar extracts. Variable inductions were observed not only between the plant extracts for a given marker gene, but also among the marker genes for a given plant extract. For instance, the eastern red cedar extract induced PR2 by 40% at the lowest dose applied, compared to an increase of 640% for KTI4 at the same dose. Likewise, PR1 expression was increased by 70% in plants treated with the highest dose of red pine extract, lower than a 200% increase observed for WRKY70 under the same treatment.

Several studies have reported the induction of PR protein-encoding and other SA-inducible genes in leaf tissue following treatment with plant extracts, as exemplified with crude extracts of seaweed42,43,44, red grape45, African lily19 or medicinal plants46. For instance, Goupil et al45. reported the local and systemic induction of SA-inducible proteins PR1 and PR2 in tobacco leaves treated with an aqueous extract of red grape, similar to Islam et al43. reporting the induction of PR1 in Arabidopsis following foliar application of a seaweed extract. Overall, our observations confirmed that ethanolic extracts from leaves or twigs of forest trees such as those considered in this study could trigger the transcription of SA-inducible genes including WRKY70 for the biosynthesis of WRKY70, a key inducer of the systemic acquired resistance (SAR) response leading to resistance against Pst DC3000 in Arabidopsis47. On a broader basis, the upregulation of SA-inducible antimicrobial proteins, such as PR2 and KTI4, suggested the potential of these extracts to promote the accumulation of defense compounds in host plant tissues eventually detrimental to biotrophic or hemibiotrophic pathogenic invaders.

RT-qPCR assays were conducted for marker genes induced by the SA antagonist jasmonic acid (JA)48 and marker genes induced under oxidative stress conditions (see Table 2) to confirm the specificity of the tree extracts in triggering SA-inducible genes induction or, on the opposite, to highlight broader gene-inducing effects eventually promoting concurrent stress-related metabolic routes (Table 4). As expected, BTH showed no inducing effect on the transcription of JA-inducible genes JAM1, LOX2, PR3 and PDF1.2 compared to control plants, even showing a marginal downregulating effect on the four genes likely explained by the well characterized antagonistic effects of SA49,50, NPR151 and WRKY7047,52 on the JA defense pathway. By contrast, the four tree extracts showed a significant inducing effect on the transcription of PR3, a gene marker for JA [and ethylene] signaling induced by different fungal and bacterial pathogens53. More specifically, extract-treated plants showed PR3 transcript levels 2 to 7 times greater than in control plants, and 3 to 10 times greater than in BTH-treated plants, at the highest dose of extract. Considering the previously reported inducing effect of JA and null effect of SA on PR3 expression54,55, these observations suggested the occurrence of additional defense gene-inducing triggers in the extracts acting along with, but independently of, the SA signaling pathway in leaf tissue. Activation of PR3 by the tree extracts was likely independent of the JA signaling pathway given the strong induction of SA-inducible genes in leaves and the mutual antagonistic relationship generally established in planta between SA and JA. Dual induction of the SA and JA signaling pathways promoting host resistance to Pst DC3000 has been observed previously in Arabidopsis leaves presenting an effector-triggered immunity hypersensitive response56 or in leaves treated with the polysaccharide elicitor chitosan57. By comparison, the weak, marginal negative effects here observed for BTH on JA-inducible genes JAM1, LOX2, PDF1.2 and PR3 suggested instead an inducing scheme independent of the SA and JA signaling pathways for PR3 protein production.

An alternative route could also explain the strong positive impact of some tree extracts on the expression of oxidative stress marker gene CAT1 (Table 4). Compared to control plants, CAT1 transcripts were increased by 3-fold to several orders of magnitude in leaves treated with the extracts of eastern hemlock, English oak or eastern red cedar. Noticeably, this inducing effect led to CAT1 transcript levels increased by more than 400 times for the cedar extract compared to control and BTH-treated plants. The basic cause of CAT1 induction remains unknown at this point but a plausible trigger could be hydrogen peroxide (H2O2), the substrate of CAT1, possibly generated in leaves upon extract treatment. Forest trees produce an array of secondary compounds to defend themselves against arthropods and pathogens28 that can not only be detrimental to microbial pathogens as shown above for Pst DC3000 (see Table 1) but also induce the production of secondary messengers like H2O2and other reactive oxygen species (ROS) involved in acclimation to abiotic stress conditions58,59. For instance, terpenoids present in large amounts in conifer (e.g., cedar) and other plant extracts show promise as potential biopesticides in agriculture for pest and pathogen control60. Considering the cell membrane-destabilizing and ROS (e.g., H2O2)-generating effects reported for terpenoids in plant extracts61, these compounds could have here triggered the induction of oxidative stress-inducible genes, such as CAT1, in tree residue-treated leaves. In support to this hypothesis, H2O2 is required for CAT1 expression in Arabidopsis leaves62, the CAT1 enzyme is readily induced by exogenous H2O2 treatment and abiotic stress cues (e.g., drought, abscisic acid, salts) that promote H2O2 production in Arabidopsis62, and the general role of stress-inducible catalases in plants is the elimination of excess H2O2accumulated in leaves under stress conditions63.

Together, our observations pointed to broad defense/stress-related gene inducing effects for the tree extracts in Arabidopsis leaves presumably involving, on the one hand, an effective activation of the SA defense pathway and, on the second hand, the induction of additional defense- or oxidative stress-inducible genes, dependent on the specific chemical composition of each extract. Numerous studies have discussed the practical potential of SA mimics like BTH and other functional analogues to activate SA-inducible genes leading to biotroph and hemibiotroph pathogen resistance in plants of economic importance64. Similarly, several studies have reported increased resistance to pathogenic fungi or bacteria in plants treated with plant extracts or products activating SA-inducible defense genes42,46,65, or in plants genetically engineered to express SAR-inducing regulators such as NPR1 or WRKY7047,66,67. As a complement, bioassays were here carried out to assess the protective potential of the four tree extracts against bacterial pathogens, again using as a model the Arabidopsis–Pst DC3000 pathosystem (Figs. 2 and 3).

Leaf senescence index for plants treated with forest tree extracts. Plants were treated with tree extracts at working concentrations of 12.5 and 25 mg mL−1, with BTH (ACTIGARD™ 50WG) at 0.5 mg mL−1 (positive control), or with sterile water used as negative control. The treatments were applied by foliar spraying (20 mL) two days before plant inoculation with Pseudomonas syringae pv. tomato DC3000 suspension. Each bar is the mean of three biological replicates (with six plants per replicate) ± se. Mean values with the same letter are not significantly different (post-ANOVA LSD, with an alpha threshold value of 5%). EH, eastern hemlock; ERC, eastern red cedar; EO, English oak; RP, red pine.

Pseudomonas syringae DC3000 populations in leaf tissue of Arabidopsis plants treated with forest tree extracts. Plants were treated with the tree extracts at working concentrations of 12.5 and 25 mg mL−1, with BTH (ACTIGARD™ 50WG) at 0.5 mg mL−1 (positive control), or with sterile water used as negative control. The treatments were applied by foliar spraying (20 mL) two days before plant inoculation with P. syringae DC3000 suspension. Each bar is the mean of three biological replicates ± se. Mean values with the same letter are not significantly different (one-way ANOVA with post-hoc LSD with an alpha threshold of 5%). EH, eastern hemlock; ERC, eastern red cedar; EO, English oak; RP, red pine.

Leaf senescence indices were first determined to rule out the possibility of phytotoxic effects for the tree extracts and to detect possible senescence-delaying effects for these extracts following bacterial infection, associated with their chemical content and/or respective impacts on ROS production and the induction of ROS-scavenging enzymes in treated leaves (Fig. 2, Supplementary Fig. S1). Plants were sprayed with the extracts two days prior to inoculation with the bacterial pathogen and their leaf senescence status assessed after 9 days, one week after infection. In brief, senescence indices for the extract-treated plants were comparable to, or lower than, the senescence index of water-treated plants (Fig. 2). Compared to the other plants, those treated with the red pine extract showed a leaf senescence index of ~ 0.55 at the lowest dose applied, about two times lower than the leaf senescence index of control plants. No such impact on leaf senescence was observed for the red pine extract on non-inoculated plants (Supplementary Fig. S1), suggesting a significant effect of this extract associated with bacterial infection. A possible explanation for this could have involved the SAR inducer WRKY70 in extract-treated plants, given the well-known antagonistic effect of this regulatory protein on leaf senescence38,68. An alternative, more likely explanation considering roughly similar induction rates for WRKY70 by the four tree extracts despite different effects on senescence, could have involved the presence of specific chemicals (e.g., terpenoids) in the red pine extract.

Plants treated with the tree extracts were then challenged with Pst DC3000 two days post-treatment to assess their overall protective effects against the pathogen and to highlight possible differential effects among the extracts given their variable toxicity against the pathogen (Table 1) and their distinct inducing effects on defense- and stress-related genes in leaves (Fig. 3). GUS expression in leaves of Pst DC3000-infected plants treated with the tree extracts was first visualized to confirm their gene inducing effects on SA-inducible defense genes (Supplementary Fig. S2), as observed above for the in vitro plantlets (see Fig. 1). Bacterial populations were then quantified one week after leaf inoculation, to evaluate the antibacterial potential of the extracts. Bacterial counts of ~ 5,000 to 50,000 colony-forming units (CFU) were observed for the extract-treated plants, similar to bacterial counts for BTH-treated plants but far lesser than the bacterial count of ~ 200,000 CFU determined for the control plants. The English oak extract, as the most toxic extract against Pst DC3000 (Table 1) but with a moderate efficiency to trigger SA-inducible marker genes compared to the eastern hemlock and red cedar extracts (Table 3), allowed for a strong, 95% decrease of CFU compared to the control treatment. The eastern hemlock extract, a potent activator of SA-inducible marker genes, also allowed for a strong decrease of CFU, similar to the effect of non-toxic SA mimic BTH, despite a weaker antibacterial effect in vitro compared to the other three extracts. Complementary studies with SA and JA pathway-compromised Arabidopsis mutants will be welcome in coming years to compare the relative impacts of extract direct toxicity and plant defense gene induction on the target pathogen, but the strong detrimental effects here observed for both the English oak and eastern hemlock extracts already suggest a dual effect for these extracts involving the two modes of action.

Several studies assessed the protective potential of plant extracts against pests and pathogens as a sustainable, eco-friendly alternative to conventional pesticides. In this study, we assessed the potential of leaf or twig extracts from four tree species of economic importance in protecting plants from bacterial infection via direct toxic effects on the target pathogen and/or indirect inducing effects on the host plant’s natural defense system. Overall, our findings confirmed the practical potential of the tree extracts as natural biopesticides for P. syringae control. They also suggested a dual antibacterial effect for these extracts, explained in different proportions from one extract to another by direct toxic effects against the pathogen and an effective induction of the host plant’s own defenses (Fig. 4).

Toxicidal (bactericidal), stress/defense gene-inducing, senescence inhibition and plant protective effects of forest tree residue extracts–A synthetic overview. MIC, minimum inhibitory concentration (•••, high toxicity; ••, moderate toxicity; •, low toxicity). MBC, minimum bactericidal concentrations (••, moderate toxicity). SA -, JA/ET- and oxidative stress marker genes (•, significant effect observed for at least one of the two tested doses; blank space, no significant induction). BTH (Actigard™ 50WG), benzothiadiazole used non-toxic SA mimic control.

Complementary studies will be welcome in forthcoming years to identify chemical(s) in the tree extracts that account(s) for the toxic effects against P. syringae and other pathogen targets. Studies will also be welcome to document possible confounding effects in the host plant responding to some extracts, as suggested by the inducing effects of the four tree extracts on JA/ET marker gene PR3 (Table 4) and the counterintuitive dose-effects observed with the red pine extract on leaf senescence inhibition (Fig. 2) and LOX2 expression (Table 4). Finally, studies will be warranted to further document the host plant’s defense response to the tree extracts. Our data pointing to the activation of SA-inducible defenses upon extract treatment might in turn indicate the establishment of a SAR response in the plant, which is known to involve the well characterized SAR master regulators WRKY70 and NPR1 and to trigger the downstream production of SA-inducible defense compounds, including several antimicrobial PR proteins. Questions remain at this point regarding the identity of (the) basic chemical trigger(s) of plant defense responses in the tree extracts, the possible importance of an oxidative stress mitigating response in leaves after extract treatment, the role of WRKY70 in the whole inducing process and the possible establishment of a SAR response at the whole plant scale. Work is underway to address these questions, and to confirm the potential of the four tree extracts for the control of economically relevant microbial diseases.

Seeds of transgenic Arabidopsis reporter line PR1::GUS were kindly provided by Prof. Corné M.J. Pieterse (Utrecht University, The Netherlands)29. The seeds were surface sterilized and placed on Murashige & Skoog (MS) medium containing 2.22 g L−1 Murashige & Skoog Basal salts (bioWorld, Dublin OH, USA), 0.5 g L−1 MES hydrate (Acros Organics, Geel, Belgium), 4 g L−1 phytagel (Alfa Aesar, Ottawa ON, Canada), and 10 g L−1 sucrose (Anachemia Inc., Montréal QC, Canada) (adjusted to pH 5.7). The Petri dishes were incubated for two days at 4 °C, and then placed vertically in a growth chamber for 14 days, to allow for the roots to grow on the surface. The plants were kept under a light intensity of 80 µmol photons m–2s–1 during the day, a light photoperiod of 16 h and a temperature regime of 23 °C during the day and 18 °C during the night. For in vivo assays, the seedlings were transplanted in 6-cell seedling starter trays containing Veranda Mix Potting Soil (Scotts Fafard, Saint-Bonaventure QC, Canada), and the plants grown on soil in the controlled growth chamber for 40 days under a 16 h light photoperiod at 21 °C, a light intensity of 210 µmol photons m–2s–1 and a relative humidity of 70%. Plants were irrigated as needed and fertilized twice a week with a 50 ppm NPK (20:20:20) solution.

Leaves of English oak, and needle-bearing twigs of eastern hemlock, eastern red cedar and red pine, were collected directly on the trees at the Jardin universitaire Roger-Van den Hende (Université Laval, Québec QC, Canada). The crude extracts were prepared as described by Delisle-Houde et al18. In brief, the tree samples were ground mechanically and macerated under agitation (100 rpm) for 24 h in 95% (v/v) ethanol at 22.5 °C. Each extract suspension was then evaporated at a temperature below 60 °C using a R-200 rotary evaporator (Büchi Labortechnik AG, Flawil, Switzerland). The resulting powder was freeze-dried and stored in the dark at room temperature in Mason jars, before eventual solubilization in sterile water for further use.

Rifampicin-resistant Pst DC3000 was used for the experiments69. The bacteria were kept in 15% (v/v) glycerol (VWR International, West Chester PA, USA) diluted in water until use, and cultivated at 28 °C on King’s B (KB) nutrient [20 g L−1 of Bacto™ Proteose Peptone No. 3 (Becton, Dickinson, and Company, Sparks MD, USA) supplemented with 1.5 g L−1 dibasic sodium phosphate (EM Science, Gibbstown NJ, USA), 10 g L−1 glycerol (VWR International), and 1.5 g L−1 magnesium sulfate (Fisher Scientific, Geel, Belgium)] solid medium [15 g L−1 of CRITERION™ Agar (Hardy Diagnostics, Santa Maria CA, USA)]. The bacteria were then inoculated in flasks containing 20 mL of KB nutrient liquid medium, allowed to grow under agitation (160 rpm) for 24 h at 28 °C, recovered by centrifugation for 5 min at 3600 × g, and finally suspended in 10 mM MgSO4 (Fisher Scientific) aqueous solution at 1 × 108 CFU mL−1. The suspension was supplemented with 0.01% (w/v) SYLGARD™ OFX-0309 (Dow Chemical Company, Midland MI, USA) for the growth chamber assays. Bacterial concentrations were monitored by measurement of optical density at 600 nm (OD600), using 0.5 McFarland standards and an Epoch 2 Microplate Spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA).

Minimum inhibitory concentrations (MICs) were determined under sterile conditions against Pst DC3000 using flat-bottom 96-well microplates (Sarstedt AG & Co., Nümbrecht, Germany). Bacterial cells (5 × 105 CFU) were suspended in 100 µL of KB liquid nutrient medium. Freeze-dried tree extracts were solubilized in sterile water, cold-sterilized with 0.22 μm filter papers and added to bacterial suspensions at the desired final concentrations (0 to 50 mg mL−1). The microplates were incubated for 24 h at 28 °C, and 10 µL of sterile 2,3,5-triphenyl-2 H-tetrazolium chloride (1 mg mL−1; Ward’s Science, Rochester NY, USA) was then added to each well70. For each plant extract, the lowest concentration at which no metabolic activity was observed (as visually inferred by the absence of red coloration in the well) corresponded to the MIC. Each concentration was tested in three replicates and the experiment was conducted twice.

Minimum bactericidal concentrations (MBCs) were determined under sterile conditions using flat-bottom 96-well microplates as described above for the MICs, except that the content of each well was spread on KB solid medium in Petri plates after 24 h incubation, instead of adding 2,3,5-triphenyl-2 H-tetrazolium chloride70. The Petri plates were incubated for 48 h at 28 °C. For each plant extract, the lowest concentration at which no growth was observed on KB solid medium corresponded to the MBC. Each concentration was tested in three replicates and the experiment was conducted twice.

Cold-sterilized tree extracts (see above) were added (5 mL) at two concentrations (12.5 and 25 mg mL−1) in Petri dishes containing 14 day-old seedlings of Arabidopsis reporter line PR1::GUS, or single leaves of 30 day-old, blooming plants of the same line. Milli-Q water and BTH (ACTIGARD™ 50WG; Syngenta Canada, Guelph, ON, Canada; 0.5 mg mL−1) dissolved in sterile water were used as negative and positive controls, respectively. Petri dishes were incubated for 48 h at 22.5 °C, and the seedlings then submitted to a GUS activity assay as described below. Quantitative RT-PCR assays were also performed to assess the activation of defense-related genes in the treated plants (see below), using DNA primers for different defense gene markers (Supplementary Table S1).

Plant samples were transferred in a microtube containing 2 mL of GUS staining solution [1 mM X-Gluc (Biosynth International, Inc., San Diego CA, USA), 100 µM phosphate buffer (Fisher Scientific, Hampton NH, USA), 10 mM EDTA (Fisher Scientific), 0.1% Triton (v/v) (Fisher Scientific), 1 mM potassium ferrocyanide (II) (Sigma-Aldrich, St. Louis MO, USA) and 1 mM potassium ferricyanide (III) (Sigma-Aldrich)] for 16 h at 37 °C. The samples were washed one time with 70% (v/v) ethanol to remove the GUS staining solution, incubated for 1 h in 70% (v/v) ethanol at 60 °C to extract chlorophyll from plant tissues, and washed three times in ethanol 70% (v/v) to remove the pigment. Leaves and seedlings were observed using a SZ2-ILST Olympus stereomicroscope (Olympus Co., Tokyo, Japan). Images were assembled using the photo merge tool of Adobe Photoshop CS6 (Adobe, Mountain View CA, USA). GUS staining was quantified using the image analysis software ImageJ as described in Béziat et al.71.

RNA extraction and RT-qPCR were performed as described in Barrada et al72. Seedlings (n = 10) of the PR1::GUS line from the in vitro assay were taken 48 h after the treatments and ground to a fine powder in liquid nitrogen. Total RNA was extracted using the Rapid Plant RNA Isolation Kit (Bio Basic, Markham ON, Canada). RNA quality was confirmed by gel electrophoresis, and genomic DNA removed by treatment with DNase I (Thermo Fisher Scientific, Waltham MA, USA). cDNA was synthesized from 500 ng RNA using a Primescript RT Reagent Kit (Takara Bio, Shiga, Japan) with random hexamer and oligo(dT) primers, and stored at -20 °C until use. Quantitative RT-PCR was performed in 1 µL of one-fifth diluted cDNA in 35-µL reactions containing the SYBR™ Green I Nucleic Acid Gel Stain (1:20,000 final dilution; Thermo Fisher Scientific), Taq DNA polymerase (1.75 units) with standard Taq buffer (New England Biolabs, Pickering ON, Canada), 0.2 mM dNTPs and 0.2 µM primers (Supplementary Table S1). The PCR reactions were performed in a LightCycler 96 instrument (Roche, Basel, Switzerland) under the following conditions: an initial denaturation cycle of 30 s at 95 °C, followed by 45 cycles of denaturation (10 s at 95 °C), annealing (30 s at 60 °C), and polymerization (30 s at 72 °C). Relative quantification of gene expression adjusted for efficiency was performed using PCR Miner73. Actin2 (ACT2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18 S-rRNA, and WRKY1 were used as reference genes. The stability of reference genes relative expression was validated according to Vandesompele et al74., with M and Cv limits of < 0.5 and < 0.25, respectively.

For the growth chamber assay, 30 day-old PR1::GUS Arabidopsis plants cultivated as described above were sprayed with the plant extract suspensions, BTH solution (20 mL per plant) or milli-Q water. Plant extracts were used at the same concentrations as for the in vitro assay above. The different formulations were applied two days before spraying the plants with a suspension of rifampicin-resistant Pst DC3000 mutant at 1 × 108 CFU mL−1. Leaf senescence was evaluated for each plant using a leaf senescence index determined according to Eq. 1. The experiment was conducted as a completely randomized design, with two replicates each including six plants (n = 12).

where the rating class corresponded to the extent of affected leaf area, based on the following score categories: 1 = less than 25%, 2 = 26–50%, 3 = 51–75%, and 4 = 76–100%.

One week after inoculation, viable bacterial (Pst DC3000) populations were determined in plant tissues according to a protocol adapted from Jacob et al75. For each treatment, three leaves of similar size and age (representing 200–300 mg) were taken randomly on three different plants in each 6-cell seedling starter tray and ground two times (30 s, in 1 mL of 10 mM MgSO4 in water), using an Omni Bead Ruptor Homogenizer (Omni International Inc., Kennesaw GA, USA). Ten microliters of each suspension were serially diluted and 10 µL of each dilution were spread on KB solid medium in Petri plates supplemented with rifampicin (Sigma-Aldrich) at 50 mg L−1. After incubation for 30 h at 28 °C, the number of CFU was counted. Populations of Pst DC3000 were expressed as CFU per milligram of fresh leaf.

Analyses of variance (ANOVA’s) and Chi-square tests were performed on the data using the R software (R-4.1.1, R Core Team, 2021, Vienna, Austria). Raw data were transformed using Tukey’s ladder of powers, and the treatments then compared using a post hoc LSD test. A non-parametric Kruskal–Wallis test (p≤ 0.05) was performed in parallel in R. The regular parametric approach was deemed valid if it gave results nearly identical to the non-parametric approach16.

All data supporting the findings of this study are available within the paper and its supplementary information files.

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Authors extend their heartfelt gratitude to Prof. Corné M.J. Pieterse (Utrecht University, Utrecht, The Netherlands) for providing seeds of transgenic Arabidopsis PR1::GUS line, and to Dr. Edel Pérez López (Université Laval, Québec QC, Canada) for providing bacterial strain Pst DC3000.

This work was supported by a grant from the Ministère de l’agriculture, des pêcheries et de l’alimentation du Québec (Programme Innov’Action Agroalimentaire), with the involvement of Investissement Québec-CRIQ and Les Fraises de l’Ile d’Orléans inc.

Département de phytologie, Faculté des sciences de l’agriculture et de l’alimentation, Université Laval, Québec, QC, G1V 0A6, Canada

Veedaa Soltaniband, Adam Barrada, Maxime Delisle-Houde, Martine Dorais, Russell J. Tweddell & Dominique Michaud

Centre de recherche et d’innovation sur les végétaux, Université Laval, Québec, QC, G1V 0A6, Canada

Veedaa Soltaniband, Adam Barrada, Maxime Delisle-Houde, Martine Dorais & Dominique Michaud

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V.S., A.B., M.D.H., R.J.T. and D.M. conceptualized the project; V.S., A.B. and M.D.H. collected the data; V.S., A.B., M.D.H.; V.S., A.B., M.D.H., R.J.T., M.D. and D.M. analyzed the data; V.S., A.B., M.D.H., R.J.T. and D.M wrote the manuscript; all authors reviewed and approved the content of the manuscript.

Correspondence to Russell J. Tweddell or Dominique Michaud.

The authors declare no competing interests.

This study was carried out with a laboratory reporter line of commonly used model plant Arabidopsis thaliana. This line was kindly provided by Prof. Corné Pieterse (Utrecht University, Utrecht 400, The Netherlands). No wild plants or endangered species of plants were used, and no field work was conducted. This study was done in compliance with relevant institutional, national, and international guidelines and legislation.

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Soltaniband, V., Barrada, A., Delisle-Houde, M. et al. Forest tree extracts induce resistance to Pseudomonas syringae pv. tomato in Arabidopsis. Sci Rep 14, 24726 (2024). https://doi.org/10.1038/s41598-024-74576-1

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Received: 12 June 2024

Accepted: 26 September 2024

Published: 21 October 2024

DOI: https://doi.org/10.1038/s41598-024-74576-1

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