Rosemary extract primes cultivar-dependent defense responses in potato against pathogen attack

Rosemary extract primes cultivar-dependent defense responses in potato against pathogen attack | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Rosemary extract primes cultivar-dependent defense responses in potato against pathogen attack Ana Paula Martin, Lucila Garcia, María Florencia Martínez, Paula Burdisso, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8399039/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Mar, 2026 Read the published version in Plant Cell Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Potato ( Solanum tuberosum L.) is a major global food crop increasingly threatened by pathogens such as Potato virus X (PVX) and Phytophthora infestans . Priming with plant extracts, including rosemary aqueous extract (ARE), provides a sustainable strategy to enhance crop immunity. Here, constitutive and ARE-induced defense responses were analyzed across four commercial cultivars: Innovator, Kennebec, Spunta, and Frital-INTA. 1 H NMR metabolomic profiling combined with defense gene expression analysis under non-infected conditions revealed cultivar-specific signatures, suggesting that basal metabolism and genetic background influence pathogen susceptibility and can be selectively tuned by ARE application. Subsequent infection assays with PVX and P. infestans validated these differential responses, identifying Innovator as more resistant and Spunta as more susceptible. Crucially, ARE pre-treatment significantly enhanced defense responses, particularly in susceptible cultivars. This priming effect resulted in a marked reduction in PVX accumulation and a decrease in P. infestans lesion size. These findings extend the established efficacy and sustainability of ARE to potato cultivation, demonstrating its capacity to act as a potent priming agent. Specifically, our results show that ARE reinforces potato immunity by integrating and amplifying both constitutive and inducible defense mechanisms, further highlighting its position as a versatile bioprotective tool for crop disease management Plant immunity Rosemary NMR metabolomics Biocontrol Priming Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key message Constitutive defense profiles differentiate potato resistance to PVX and . Aqueous rosemary extract (ARE) primes susceptible cultivars, offering a sustainable strategy to boost resilience against these major foliar pathogens. Introduction Potato ( Solanum tuberosum L.) plays a crucial role in global agriculture due to its phenotypic plasticity and adaptability to diverse environments. With over 4,000 varieties, primarily originating from the Andes, and an annual production exceeding 350 million tons, potatoes rank among the top crops alongside maize, wheat, and rice (Devaux et al. 2019 ). Supporting the subsistence of over 1 billion people, potatoes provide a vital source of carbohydrates, proteins, dietary fibers and micronutrients (Navarre et al. 2019 pás et al. 2024 ). Their skin contributes essential vitamins and minerals that enhance the overall nutritional value (Dessì et al. 2025 ). Most commercial potatoes belong to autotetraploid cultivars of Solanum tuberosum subsp. tuberosum L, which displays remarkable diversity in traits such as phenological maturity, yield, and tuber characteristics including shape, size, and skin color (Tagliotti et al. 2018 ; Bradshaw 2022 ). Their versatility is further highlighted by the wide range of phytochemical profiles and the existence of market classes adapted for fresh consumption, processing, or seed production (Navarre et al. 2019 ). Despite advances in breeding and genomics, potato production continues to face major challenges, including adverse environmental conditions, climate change, and pathogen pressures (Tiwari et al. 2022b , a ). Climate-driven changes in temperature, humidity, and precipitation are reshaping plant–pathogen interactions worldwide, influencing pathogen distribution, virulence, and epidemic dynamics (Garrett et al. 2022 ). Among the most devastating pathogens, Phytophthora infestans (Mont.) de Bary, the causal agent of late blight, has shown remarkable adaptability not only to fluctuating temperatures but also to fungicide pressure, resulting in extended epidemic potential under warming scenarios (Wu et al. 2022 ). This oomycete remains a major constraint for potato production globally, capable of causing yield losses of 70–80% under conducive environmental conditions (Fry 2016 ; Savary et al. 2019 ). Viral diseases such as Potato virus X (PVX) substantially contribute to global potato yield losses, particularly under co-infections with potyviruses and luteoviruses, where reductions of up to 80% have been reported (Pacheco et al. 2012 ; Nicaise 2014 ; Syller and Grupa 2016 ; Verchot 2022 ). Although resistance to PVX and P. infestans has been previously characterized in commercial cultivars under field conditions, it remains unclear whether these resistance traits persist under current environmental and agronomic pressures. Therefore, developing effective and sustainable disease management strategies to maintain crop resilience and productivity remain major challenges. In this framework, natural plant extracts have emerged as promising bioprotectants against pathogens, capable of triggering defense priming and enhancing immunity without the metabolic cost of constitutive resistance (Kerchev et al. 2020 ; Aremu et al. 2024 ). For instance, rosemary ( Salvia rosmarinus Spenn) aqueous extracts (ARE), rich in phenolics such as rosmarinic acid (RA), have been shown to induce plant defense responses against a broad range of pathogens, including virus, bacteria, and fungi (Martin et al. 2023 ). Plant immunity relies on a multilayered defense system integrating constitutive and inducible responses. Preformed antimicrobial metabolites and structural barriers provide basal protection (Osbourn 1996 ; Vleeshouwers et al. 2000 ; Navarre and Mayo 2004 ; Halim et al. 2007 ; Favaro et al. 2020 ). Upon pathogen perception, pattern-recognition receptors (PRRs) and intracellular resistance (R) proteins, most of which belong to the nucleotide-binding leucine-rich repeat (NLR) family (van Wersch et al. 2020 ; Yu et al. 2024 ; Coban et al. 2025 ), activate defense signaling cascades that culminate in a hypersensitive response (HR) characterized by programmed cell death that restricts pathogen spread (Greenberg and Yao 2004 ; Balint-Kurti 2019 ). In this study, we analyzed the constitutive defense responses of four commercially potato cultivars -Innovator, Kennebec, Spunta and Frital-INTA - grown in major potato-producing regions of Argentina (Mondino, M.C.; Grasso, R.; Balaban, D.; Ortiz Mackinson, M.; Cardozo, F.; Timoni, R.; Vita Larrieu 2021). Metabolomic and molecular analyses performed on non-inoculated leaves revealed distinct basal defense profiles among cultivars. Innovator showed higher accumulation of defense-related metabolites and stronger expression of defense-associated genes, consistent with its resistance phenotype, whereas Spunta exhibited a weak basal defense signature indicative of high susceptibility to both pathogens (Nyalugwe et al. 2012 ; Armstrong et al. 2019 ). Controlled inoculation assays with Potato virus X (PVX) and P. infestans confirmed these contrasting responses. Based on these results, Spunta was selected as a model to evaluate priming strategies aimed at enhancing defense activation under field-relevant conditions. ARE, applied prior to pathogen inoculation, significantly reduced PVX accumulation and late blight lesion severity, reinforcing defense responses through a priming mechanism in this susceptible genotype. Our findings demonstrate that cultivar-specific metabolomic and transcriptional signatures underlie resistance to PVX and P. infestans , and that ARE-mediated priming effectively enhances potato immunity. This approach represents a sustainable strategy to strengthen crop resilience against foliar pathogens under changing climatic conditions. Materials and Methods Germplasm and clonal propagation of potato cultivars Tubers of Solanum tuberosum L. cultivars Spunta, Kennebec, Innovator and Frital-INTA (hereafter referred to as Frital) were obtained from potato growers across the main horticultural area of Rosario, Argentina, extending roughly between 33°S and 60°W (Mondino, M.C.; Grasso, R.; Balaban, D.; Ortiz Mackinson, M.; Cardozo, F.; Timoni, R.; Vita Larrieu 2021). Cultivar identity was confirmed by specialists from National Agricultural Technology Institute (INTA), using molecular markers (Ghislain et al. 2009 ). The cultivars used in this study are listed in Table S1 , together with information on their parentage and previously reported resistance ( R ) genes associated with the pathogens analyzed herein. Tubers harvested from mature plants were stored at 4°C until sprouting. Once sprouts developed, they were transferred to soil in controlled-environment chambers for clonal propagation, following the methods described in (Sánchez et al. 2010 ). To obtain physiologically uniform and tender tissues particularly suitable for pathogen infection, metabolomic and gene expression analysis, the plants were then transferred to tissue culture. Internodal stem sections were excised, surface-sterilized by gentle washing with a bleach solution followed by sterile water, and placed horizontally in sterile containers containing 0.5 × Murashige and Skoog (MS) medium (Vollmer et al. 2021 ). Subcultures were performed every four weeks to generate sufficient plantlets, which were subsequently transferred back to soil for pathogen infection assays and molecular analyses. The cultivar Pentland Ivory, carrying the Nb resistance gene in the simplex condition ( Nb nb nb nb ), was self-pollinated to obtain resistant ( Nb ) and susceptible ( nb ) progeny (Marano et al. 2002 ). These progenies were clonally propagated by rooting apical internode cuttings, as described by Sánchez et al. 2010 . Pathogen-free potato plants were maintained in a growth chamber at 20 to 25°C with 60% relative humidity, a 16 h photoperiod and a light intensity of 150 to 200 µE m –2 s –1 . PVX cDNA clones and viral inoculation The pPVX204 plasmid (Baulcombe et al. 1995 ), containing the PVX UK3 full-length cDNA fused to the green fluorescent protein ( GFP ) gene, was modified to create the PVX ROTH1 strain expressing GFP (PVX ROTH1-GFP). This was accomplished by replacing the DNA fragment positioned between the unique BamH I and Apa I restriction sites in PVX-GFP with the corresponding fragment from the PVX ROTH1 plasmid (Malcuit et al. 1999 ), which includes the 5’ terminal region of the gene encoding the 25 kDa movement protein (Fig. S1 A). The resulting plasmid was designated PVX ROTH1. The functionality of the PVX ROTH1-GFP infectious clone was evaluated by agroinfiltration in Nicotiana benthamiana leaves using mechanical inoculation. For each plant, 10 µg of plasmid DNA (pPVX ROTH1-GFP) was applied in a phosphate buffer (10 mM, pH 7.2) using gentle abrasion. At 10 days post-inoculation (dpi), successful infection was confirmed by the presence of green fluorescence in symptomatic leaves under ultraviolet (UV) illumination, in addition to characteristic PVX-induced symptoms such as mosaic and leaf curling observed in systemic tissues (Fig. S1 B). A homogenate inoculum containing infectious virions was prepared from PVX ROTH1-GFP infected N. benthamiana leaves, following the protocol described by Garcia et al. 2023 . Crude sap was adjusted to a concentration of approximately 0.5-1 infective virions/µL and used to inoculate different potato cultivars (Malcuit et al. 1999 ). A randomized experimental design was employed, including eight to ten biological replicates per cultivar. Plants were maintained under controlled environmental conditions, as described previously. Control plants were mock-inoculated with potassium phosphate buffer (10 mM, pH 7.2) only. The progression of local infection was monitored over a 14-day period using a stereomicroscope (BH2; Olympus, Tokyo, Japan). Agrobacterium- mediated transient expression. Potato plants at the 4–5 leaf stage were inoculated by pressure infiltration with Agrobacterium tumefaciens strain C58C1, transformed with pBIN25K1 or pBIN25K3 plasmids (Sánchez et al. 2010 ). Agrobacterium cells were cultured in 5 ml of L medium (Sambrook, J.; Fritsch, E.F.; Maniatis 1989 ), supplemented with kanamycin (50 µg/mL) and tetracycline (5 µg/mL), and incubated at 28°C to saturation for 16 h. After centrifugation at 750 × g, cells were resuspended in infiltration buffer containing 10 mM MgCl 2 , 10 mM morpholineethanesulfonic acid (MES, pH 5.6), and 100 µM acetosyringone (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), and the optical density (OD 600 ) was adjusted to 0.8. The cultures were incubated in dark at room temperature for 3 hours with gentle shaking prior to agroinfiltration. Plants were monitored for up to 3 days at 22°C and 50–60% relative humidity. Zoospore inoculum preparation, infection assays, and lesion quantification of P. infestans The P. infestans isolate PiNSL-19, classified as genotype EU_2_A1 genotype (Juárez, M.; Azcue, J.; Cano Mogrovejo, L.; Lamour, K.; Bravo-Almonacid, F.F.; Lucca, M.F.; Segretin 2024), was originally obtained from symptomatic cultivar Spunta tubers collected in San Luis Province, Argentina. Cultures were maintained on rye sucrose agar (RSA) medium, prepared according to (Van West et al. 1998 ). Briefly, 60 g of rye grains were surface-sterilized in 0.25% sodium hypochlorite for 4 min, rinsed, and incubated at 25°C for 24 h to promote germination. The grains were then dried at 55°C for 3 h, ground, and used to prepare RSA medium by adding 20 g of sucrose and 15 g of agar to a final volume of 1 L. The medium was autoclaved, and cultures were initiated from mycelial discs cryopreserved in 5% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen (PW 1988). Active cultures were maintained at 4°C and subculture weekly. For zoospore production, P. infestans was grown on RSA plates at 18°C in the dark for 11–14 days (Van West et al. 1998 ). Sporulating cultures were flooded with 3–5 mL of chilled sterile water and incubated at 4°C for 1–2 h to stimulate zoospore release. Zoospore concentrations were monitored every 30 min until reaching the concentration of use (2×10⁴ − 3×10⁴ zoospores/mL). The suspension was then transferred to microcentrifuge tubes and maintained at 4°C during inoculation procedures. In vitro infection assays were conducted using plantlets of potato cultivars Innovator, Kennebec, Spunta and Frital, grown on MS medium supplemented with 2% (w/v) sucrose. A 10 µL droplet of zoospore suspension was applied to the leaf surface, following the protocol of (Huang et al. 2005 ). Disease progression was documented at 5 dpi via digital imaging. Detached leaf assays were performed on leaves from 4-to 5-week-old soil-grown plants of potato cultivars. Plant growth and infection assays were conducted in parallel at two independent laboratories, INGEBI-CONICET (Buenos Aires) and IBR-CONICET (Rosario). In both facilities, plants were cultivated in growth chambers under standardized conditions (24°C, 16 h light/8 h dark photoperiod, 60% relative humidity). Experimental procedures were independently replicated at both sites to ensure reproducibility. The two youngest fully expanded compound leaves were detached and placed abaxial side up on water-statured floral foam blocks arranged in plastic trays. Each leaflet was then inoculated on the abaxial surface with a 10 µL drop of zoospore suspension. Control leaflets were inoculated with sterile water. After inoculation, the trays were sealed with transparent plastic film to maintain high humidity and prevent desiccation. The inoculated leaves were incubated under the same controlled growth chamber conditions described above. Lesion development was assessed up to 5 dpi, following the protocol by Gabriel et al. 2007 . Both adaxial and abaxial leaf surfaces were photographed under visible light, and the abaxial surface was additionally photographed under UV light (365 nm) to visualize necrotic areas. Lesion area was quantified using ImageJ software by adjusting brightness, contrast and color saturation parameters with the “Adjust-Color Threshold” function, following a protocol adapted from the UF IFAS Horticultural Crop Physiology Lab (Agehara et al. 2020 ). Leaf and lesion areas were measured for each leaflet. In cases of overlapping lesions, the midrib was used as a boundary to define lesion margins. Proportion of lesion area was calculated as the ratio of lesion area and total leaf area. Rosemary extract preparation, plant treatment and pathogen inoculation Lyophilized aqueous rosemary extract (ARE) was prepared as described by Martin et al. 2023 . The extract was standardized to a final rosmarinic acid (RA) concentration of approximately 400 µM, and all treatments were quantified based on RA content. Although RA is the major phenolic component, ARE displays stronger protective activity than RA alone, suggesting the contribution of additional bioactive compounds (Martin et al. 2023 ). Four-week-old soil-grown potato plants were treated by spraying 100 µL of ARE onto the adaxial surface of one fully expanded leaf per plant, once daily for two consecutive days. Control plants were similarly treated with water. Pathogen inoculation was performed 24 h after the second application (Fig. S2). Lesion area associated to P. infestans infection was quantified using Image J as indicated above. Data were modeled using a beta distribution within a randomized block design framework, suitable for proportion data derived from normally distributed variables. The experiment day was treated as a random variable. Statistical analyses were performed in RStudio (R version 4.0.1) using ggplot2 and RColorBrewer for visualization, glmmTMB for Beta model fitting, DHARMa for model assumption checks, and emmeans for post-hoc comparisons. Sample preparation for proton nuclear magnetic resonance (H NMR) spectroscopy Leaf samples were prepared for metabolite analysis following the protocol described by Kim et al. 2010 . A total of 20 leaf extract samples (five per cultivar) were analyzed. Apical leaves (~ 200 mg) from plants at the 3–4 leaf stage were collected, excluding petioles, snap-frozen in liquid nitrogen, and homogenized using a tissue disruptor (MM-400, Restch GmbH, Haan, Germany). Homogenates were lyophilized, and ~ 30 mg of the resulting dry powder was subjected to polar metabolite extraction using 750 µL of phosphate buffer (90 mM, pH 6.0) prepared in deuterium oxide (D 2 O) and containing 0.1% (w/v) 3-(trimethylsilyl)-2,2,3,3-d4-propionic acid (TSP) as an internal standard. Samples were vortexed for 2 min at room temperature and sonicated three times (5 min each, with alternating 30 s on/off pulses) in an ultrasonic bath (Branson SS10 EMT, Brookfield, Connecticut, USA). Following centrifugation at 25,000 × g for 30 min at 4°C, 500 µL of clarified supernatant was transferred to 5 mm NMR tubes (Wilmad NMR, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) for spectral analysis. This methodology allows to simultaneously detect primary (sugars, organic acids, amino acids) and secondary metabolites (flavonoids, alkaloids, terpenoids), providing quantitative and structural information. 1 H NMR spectra were obtained at 300 K on a Bruker Avance III 700-MHz NMR spectrometer (Bruker Biospin, Rheinstetten, Germany) equipped with a 5-mm TXI probe. One-dimensional 1 H NMR spectra of leaf extract were acquired using a standard 1-D NOESY pulse sequence (noesygppr1d) with water presaturation. The mixing time was set to 10 ms, the acquisition time to 2.228 s, and the relaxation delay to 4 s. Spectra were acquired using four dummy scans and 32 acquisition scans, with 64 K time domain points and a spectral width of 20 ppm. Free induction decays were multiplied by an exponential window function with a line broadening factor of 0.3 Hz prior to Fourier transformation. Data pre-processing and multivariate analysis of H NMR spectra Spectroscopic data were processed in MATLAB (version R2015b, The MathWorks). Spectra were referenced to TSP at 0.0 ppm, followed by baseline correction and phase adjustment using custom functions developed at Imperial College London (provided by T. Ebbels and H. Keun). Each spectrum was segmented into integrated regions (“bins”) of equal width (0.04 ppm, standard bucket width). Non-informative regions containing no metabolite signals, including the TSP signal and the water signal resonance region (between 4.9 and 4.6 ppm), were excluded. Spectra were normalized using the probabilistic quotient (PQN) method (Dieterle et al. 2006 ). Alignment was performed using the recursive segment-wise peak alignment (RSPA) algorithm within user-defined spectral windows (Veselkov et al. 2009 ). Spectral data matrices were subjected to multivariate curve resolution-alternating least squares (MCR-ALS) in MATLAB®, with spectra segmented into windows containing relevant signals, and non-negativity constraints were applied. The optimal number of components was determined, and integrated signals were exported for further matrix construction. The resulting data matrix was imported into MetaboAnalyst 6.0 software (Pang et al. 2024 ) for multivariate statistical analysis (Table S2). Data were centered and scaled using unit variance (UV). Principal component analysis (PCA) was performed to explore sample distribution, and orthogonal partial least squares discriminant analysis (OPLS-DA) was applied to maximize group separation and identify discriminant signals. Supervised models were validated using 200 permutation tests. Discriminant variables, showing significant differences, were identified through a Volcano plot, applying a fold change threshold of 2 and a significance level of p < 0.05. Only the discriminant signals were subsequently assigned, ensuring that the interpretation focused exclusively on the features that contributed meaningfully to group separation. Discriminant signals were annotated using the Biological Magnetic Resonance Data Bank (BMRB) and the Human Metabolome Database (HMDB) (Wishart et al. 2013 ). RNA extraction, reverse transcription, and quantitative PCR (qRT-PCR) Total RNA was extracted from three leaf disks (~ 100 mg of fresh tissue) per cultivar using the NucleoSpin® RNA kit (Macherey-Nagel, Düren, Germany), according to the manufacturer instructions. Reverse transcription was performed with RevertAid (ThermoFisher Scientific, Waltham, Massachusetts, USA). For virus quantification, 1 ng of DNase-treated RNA was used, whereas 1 µg of total RNA was employed for the analysis of plant gene expression. In both cases, oligo (dT) 12–18 primers were used to synthesize first-strand complementary DNA (cDNA). Quantitative PCR (qPCR) reactions were carried out in a final volume of 20 µL, containing 1 µL of EvaGreen dye (Biotium, Fremont, California, USA), 5–10 pmol of each primer, 3 mM MgCl₂, cDNA (1:10 dilution), and 1 U Taq DNA polymerase (PBL, Buenos Aires, Argentina). Amplification was monitored using the Mastercycler® ep realplex system (Eppendorf, Hamburg, Germany). Primer sequences targeting the PVX ROTH1 coat protein ( CP ) and plant defense-related genes, including Phenylalanine Ammonia-Lyase ( PAL ), Isochorismate Synthase 1 ( ICS1 ), Pathogenesis-Related Protein 1 ( PR1 ), UDP-glucosyltransferase 74B1 (UGT74B1 ), Allene Oxidase Synthase 2 ( AOS2 ), Acyl-coenzyme A oxidase (ACX1) , Acyl-coenzyme A oxidase (ACX3) , and Elongation Factor 1-α ( EF-α ), as well as thermal cycling conditions are provided in Table S3. Relative transcript levels were calculated using the ΔΔCt method (Livak and Schmittgen 2001 ). Gene expression data were normalized against EF-α transcript levels. For viral CP RNA quantification, samples taken at 24 hours post inoculation (hpi) were used as reference. For plant gene expression analysis in non-inoculated plants, samples from cultivar Spunta served as reference. In the priming experiments, PVX ROTH1 CP RNA and plant defense marker genes were analyzed at 24 and 48 hpi. Water treated plants at each time served as reference. Each experiment included four biological replicates per cultivar and, each comprising pool material from two to four leaves collected from independent plants. Assays were independently repeated three times. Statistical significance was performed using one-way ANOVA ( p < 0.05). Results Basal metabolic profiles of potato cultivars to evaluate intrinsic defense capacity Constitutive metabolic traits can strongly influence how plants respond to environmental challenges and pathogen attacks, ultimately shaping their resilience under changing climatic conditions (Salam et al. 2023 ). To investigate whether such traits underlie differences in susceptibility to PVX and P. infestans , leaves from the cultivars Innovator, Kennebec, Spunta, and Frital were analyzed using ¹H NMR spectroscopy. This approach established metabolic baselines for each cultivar, providing a framework to assess constitutive defense capacity and the potential impact of defense-priming strategies in susceptible genotypes. Across the four cultivars, 270 metabolic features were detected, with 197 assigned to known metabolites. Among these, 11 metabolites were annotated and 9 identified as discriminant variables, including the alkaloid trigonelline, the amino acid asparagine, the indole derivative 3-indolylmethyl, and the fatty acid linoleic acid (Table S2). Principal component analysis (PCA) of the 197 integrated ¹H NMR peaks explained 63.2% of the total variance, with PC1-PC4 contributing 27.2%, 15.1%, 11.8%, and 9.1%, respectively. As shown in Fig. 1 a, PC1 and PC3 clearly separated Innovator from the other cultivars, reflecting its distinct constitutive metabolic profile associated with resistance. PC2 and PC4 distinguished Kennebec and Spunta, whereas Frital overlapped with both susceptible cultivars. The obtained metabolic profiles align with previously reported resistance loci (Table S1 ): Innovator carries multiple Rpi-R genes and the Nb gene, consistent with resistance to P. infestans and PVX strain ROTH1 (PVX ROTH1), respectively. In contrast, both Kennebec and Spunta lack Nb and contain only Rpi-R1 , which confers limited or no protection against virulent P. infestans isolates carrying non-recognized Avr1 variants (Coomber et al. 2024 ). Consequently, both cultivars are likely susceptible to PVX ROTH1 and P. infestans . Frital presents a less defined genetic background, suggesting a potential intermediate or susceptible phenotype. Based on these genetic backgrounds, we categorized the cultivars into putative resistant- or susceptible-like groups. To investigate metabolic differences between potentially resistant- and susceptible-like cultivars, supervised OPLS-DA was performed comparing Innovator with Kennebec and Spunta, while Frital was excluded from subsequent binary analysis. This approach revealed clear separation between the two groups (Fig. 1 b). A heat map of the 30 most abundant metabolites across five biological replicates per cultivar highlighted specific metabolic signatures and facilitated direct comparison of metabolite abundance between the two groups (Fig. 1 c). Differential metabolite accumulation between resistant- and susceptible- groups was further assessed using volcano plot analysis (fold change > 2.0; p < 0.01), which identified metabolites with statistically significant differences (Fig. 1 d). In the resistant-like group, 3-indolylmethyl, trigonelline, linoleic acid (or a related derivative), and two unidentified signals (U_32_1 and U_87_2) were upregulated, whereas asparagine and three unidentified signals (U_33_4, U_38_2, and U_115_1) were downregulated. Interestingly, among the susceptible-like cultivars, Spunta showed the lowest metabolic abundance (Fig. S3a). Taken together, untargeted NMR analyses revealed cultivar-specific metabolic signatures. Innovator was enriched in trigonelline, linoleic acid and indole-derived metabolites, reflecting a constitutive metabolic state that may support basal pathogen defense. In contrast, Kennebec and Spunta displayed a narrower and less diverse metabolic profile, particularly in compounds potentially associated with basal defense, suggesting a metabolic context less favorable for early stress responses. Constitutive expression of defense-related genes in potato cultivars Basal defense responses were also assessed by quantifying the expression of four candidate defense genes in non-inoculated leaves of Innovator and Spunta, selected as representative cultivars with contrasting resistance loci and constitutive metabolic profiles identified in the metabolomic analysis. These genes were PAL1 , encoding phenylalanine ammonia-lyase, a pivotal enzyme in phenylpropanoid biosynthesis; ICS , encoding isochorismate synthase, essential for salicylic acid (SA) production; PR1 , a canonical SA-responsive pathogenesis-related gene; and UGT74B1 , a putative UDP-glycosyltransferase involved in indolic glucosinolate glycosylation (Vleeshouwers et al. 2000 ; Navarre and Mayo 2004 ; Grubb et al. 2004 ). Notably, PR1 expression was markedly elevated in Innovator (19.52 ± 10.71 vs. 1.01 ± 1.13), a widely used marker of SA-dependent defense signaling, while UGT74B1 showed a moderate but consistent increase (3.06 ± 0.63 vs. 1.04 ± 0.34). In contrast, PAL1 and ICS expression levels were comparable between cultivars (Fig. S3b). These patterns, together with elevated tryptophan-derived metabolites in Innovator, indicate a constitutive defense state that may contribute to its enhanced pathogen resistance. Cultivar-specific potato defenses against PVX ROTH1 and P. infestans identify candidates for ARE priming Integrated infection assays were performed to validate the predicted resistance/susceptibility profiles and to investigate how constitutive metabolic and transcriptional signatures shape cultivar-specific defense. Experiments were accomplished using PVX ROTH1 and a locally isolated P. infestans strain. This experimental framework provides evidence to identify and confirm susceptible cultivars suitable for defense reinforcement via ARE-mediated priming. To characterize antiviral responses, leaves from each cultivar (Innovator, Frital, Kennebec, and Spunta) were mechanically inoculated with a GFP-expressing PVX ROTH1 clone (PVX ROTH1-GFP). Pentland Ivory, which carries the Nb resistance gene, and a susceptible nb genotype derived from selfing were included as controls for PVX resistance and susceptibility, respectively (Malcuit et al. 1999 ; Marano et al. 2002 ). Fluorescence imaging revealed restricted viral replication in Innovator and Frital, where GFP signals were confined to the inoculation site and associated with localized hypersensitive response (HR) lesions. This pattern is consistent with Nb -mediated recognition of the viral PVX 25-kDa movement protein (25K1) (Fig. 2 a). In contrast, Kennebec and Spunta exhibited extensive GFP fluorescence spreading beyond the inoculation site, reflecting a susceptible phenotype with unrestrained viral progression. Quantification of PVX CP RNA at 48 hpi corroborated these observations: Innovator and Frital showed CP transcript levels of 1.23 ± 0.44- and 1.57 ± 0.51-fold relative to the resistant Nb control, respectively. Conversely, Kennebec and Spunta exhibited significant increases of 10.84 ± 2.63- and 18.17 ± 7.36-fold, respectively, while the susceptible nb control displayed the highest accumulation (36.3 ± 13.7-fold relative to Nb ) (Fig. 2 b). The Nb -mediated resistance was subsequently confirmed by agroinfiltration assays expressing either the 25K1 effector or the homologous 25K3 from PVX UK3. At 72 hpi, Innovator and Frital developed localized HR upon 25K1 expression—mirroring the Nb -positive control—whereas no HR was observed in Kennebec, Spunta, or any cultivar expressing 25K3 (Fig. S4a). Genotyping with SPUD237 and SPUD839 markers further verified the presence of the Nb gene in Innovator and Frital and its absence in Kennebec and Spunta (Fig. S4b). Collectively, these results demonstrate for the first time that Frital possesses resistance to PVX ROTH1 similar to Innovator, while Kennebec and Spunta exhibit a susceptible phenotype. To evaluate cultivar-specific defense against oomycetes, in vitro propagated plants were inoculated with P. infestans isolate PiNSL-19. Innovator developed localized HR lesions that restricted pathogen spread and sporulation, whereas Spunta and Kennebec showed severe disease symptoms. Frital exhibited an intermediate phenotype, with lesion development falling between the resistant and susceptible cultivars. These responses were recapitulated in detached leaves from soil-grown plants: Innovator restricted lesion expansion, while Spunta and Kennebec developed large, sporulating lesions. Lesion areas were quantitatively assessed at 4 dpi and normalized to Spunta (Fig. 3 a,b). Together, these results confirm that cultivar-specific responses to PVX and P. infestans are consistent across infection assays, reflecting the combined action of basal and effector-triggered immunity. The strong correlation between molecular markers and observed phenotypes supports the use of genetic screening to streamline the identification of susceptible cultivars. Consequently, this approach integrates constitutive and inducible defense mechanisms to target cultivars for ARE-mediated resistance reinforcement under climate-driven pathogen pressure. ARE treatment primes potato defense responses against viral and oomycete pathogens Previous studies demonstrated that ARE functions as an effective priming agent in diverse plant species, including N. tabacum , Glycine max , and Citrus limon . This treatment enhances resistance against a wide range of pathogens, including viruses, bacteria and fungi, by reinforcing basal immune signaling and promoting a faster activation of inducible defense pathways upon infection (Martin et al. 2023 ). To evaluate the potential of ARE to prime inducible defenses against both PVX and P. infestans , the highly susceptible cultivar Spunta, was selected. This genotype, which lacks effective genetic resistance mechanisms to multiple pathogens, provides an appropriate model to assess the efficacy of ARE-mediated priming. In parallel, the PVX ROTH1-resistant cultivar P. Ivory (carrying the Nb gene) and the susceptible nb genotype were included as controls, allowing comparison between ARE-induced responses and those conferred by genetic resistance. Plants were treated twice with 100 µL of ARE at 24 h intervals, and 24 h after the last application, leaves were inoculated with PVX ROTH1-GFP, and local infection symptoms were monitored for a 10-day period. Viral movement and replication were monitored by fluorescence microscopy and qPCR analysis of CP -PVX levels (Fig. 4 a,b). In Spunta, ARE treatment restricted viral cell-to-cell movement and significantly reduced viral accumulation compared to water-treated controls at 24 and 48 hpi (Fig. 4 a,b). A similar decrease in CP -PVX transcription levels was observed in the PVX ROTH1-susceptible cultivar P. Ivory, indicating that both Spunta and this genotype responded comparably to ARE treatment in the absence of functional Nb resistance gen. qPCR analysis showed an 80–90% reduction in CP -PVX transcript levels at 24 and 48 hpi in ARE-treated susceptible cultivars. In contrast, in the resistant Nb genotype, which inherently restricts PVX ROTH1 accumulation through HR, ARE treatment further reduced fluorescence intensity at 48 hpi, suggesting an additive priming effect. Fluorescence was only observed in the area surrounding the initial infection site to accurately assess viral spread within the tissue (Fig. 4 a). Accordingly, CP -PVX transcript levels remained consistently low regardless of treatment, likely due to the strong immune response mediated by Nb -dependent resistance (Fig. 4 b). Together, these results demonstrate that ARE limits PVX ROTH1 replication in susceptible cultivars and can further potentiate antiviral defenses even in the presence of functional resistance genes. Based on prior studies in N. tabacum infected with tobacco necrosis virus strain A (TNVA), where ARE treatment enhanced SA accumulation and defense gene expression (Martin et al. 2023 ), we hypothesized that ARE may similarly prime SA-mediated defenses in potato against PVX ROTH1. To test this, we analyzed the expression of key SA-related genes involved in biosynthesis ( PAL, ICS1 ) and downstream signaling ( PR1 ). In Spunta, ARE treatment induced a delayed upregulation of ICS1 transcripts at 48 hpi, without significantly modulation of PAL or PR1 expression, suggesting a temporally limited activation of the isochorismate- dependent SA biosynthetic pathway (Fig. 4 c). For comparison, the control cultivar Pentland Ivory, carrying either the resistant ( Nb ) or susceptible ( nb ) alleles, was also analyzed. In these control genotypes, ARE treatment triggered earlier and substantially stronger transcriptional activation of SA-associated genes. Specifically, PAL and PR1 , were significantly upregulated at 24 and 48 hpi in both genotypes. Interestingly, ARE-treated nb plants reached PAL and PR1 expression levels comparable to those of the resistant Nb genotype, suggesting that ARE can enhance SA-dependent defense activation even in the absence of a functional Nb resistance gene. This differential response, stronger in Pentland Ivory than in Spunta, highlights a genotype-dependent predisposition to ARE-mediated priming, whereby certain genetic backgrounds may exhibit an enhanced capacity to activate defense signaling independently of effector-specific recognition. Figure 4 . Aqueous rosemary extract (ARE) confers protection against PVX ROTH1-GFP in potato cultivars. a. Potato leaves of cultivars Pentland Ivory ( Nb and nb ) and Spunta (Sp) were treated with aqueous rosemary extract (ARE) or water, and then inoculated with PVX ROTH1-GFP. Leaves were imaged by laser scanning confocal microscopy at 2 days post-inoculation (dpi) in resistant and 4 dpi in susceptible cultivars. b. Reverse transcription and quantitative PCR (qPCR) analysis at 24 and 48 h post-inoculation (hpi) in PVX ROTH1-GFP-inoculated plants treated with ARE or water. Values were normalized to the constitutive EF-α gene and fold changes were expressed relative to inoculated water-treated leaves (controls) of Pentland Ivory nb genotype ( nb ) for each time. Mean ± standard deviation (n = 4 replicates, each consisting of four leaves from four different plants) are shown. Different letters indicate significant differences at p < 0.05 (one-way ANOVA, Tukey’s test). c. qPCR analysis of PAL , IC 1, and PR1 at 24 and 48 hpi with PVX ROTH1-GFP in potato cultivars treated with ARE or water. Values were normalized to the constitutive EF-α gene and fold changes were expressed relative to inoculated water-treated leaves (control) at each time. Mean ± standard deviation of 5 replicates, each consisting of five leaves from five different plants are shown. Asterisks indicate significant differences at * p < 0.05, ** p = 0.008 (one-way ANOVA, Tukey’s test). EF-α , ELONGATION FACTOR 1-α ; PAL , PHENYLALANINE AMMONIA-LYASE ; ICS , ISOCHORISMATE SYNTHASE ; PR1 , PATHOGENESIS-RELATED PROTEIN 1 . For P. infestans infection assays, potato plants of the highly susceptible cultivar Spunta were treated twice with 100 µL of ARE. Twenty-four hours after the second application, detached leaves were inoculated with the virulent isolate PiNSL19 (Fig. S2). Disease progression was monitored over 5-days based on lesion development and expansion. At 72 hpi, ARE-treated leaves showed smaller and lighter necrotic lesions compared with the larger, darker ones in water-treated controls (Fig. 5 a and Fig. S5a). Quantitative image analysis indicated a 30% reduction in lesion size and overall disease severity ( P < 0.05; Fig. 5 b and Fig. S5b). In vitro diffusion assays confirmed that ARE had no direct effect on P. infestans mycelial growth, suggesting that protection was associated with activation of host defenses rather than antifungal activity ( data not shown ). Defense against P. infestans relies on coordinated hormonal regulation, with SA responses dominating the early biotrophic phase and jasmonic acid (JA) contributing during the necrotrophic stage (Halim et al. 2007 ; Zhang et al. 2023 , 2025 ). To explore whether ARE affected these pathways, we analyzed the expression of representative marker genes (Fig. 5 c). All three SA-associated genes were induced at 72 hpi, but no significant differences were detected between ARE- and water-treated plants, indicating that ARE does not substantially enhance SA-mediated defenses under the tested conditions. For the JA pathway, AOS2 involved in early JA biosynthesis, and ACX1 and ACX3 , encoding acyl-CoA oxidases of the β-oxidation pathway, were selected based on previous transcriptomic data from Spunta infected with PiNSL19 (Juarez 2024 ; Juarez et al. 2024 ). AOS2 expression increased at 48 hpi in both treatments, whereas ACX1 and ACX3 transcripts were consistently lower in ARE-treated plants (Fig. 5 c). These findings indicate that ARE enhances Spunta resistance to P. infestans , accompanied by moderate activation of SA- and JA-related genes. The magnitude and timing of these responses suggest that additional, genotype-dependent regulatory pathways may also contribute to the observed protection. Discussion Understanding how potato cultivars respond to pathogens under fluctuating environmental conditions is critical, as genetic resistance is often incomplete or can be overcome by emerging threats. ARE, a plant-derived extract with broad-spectrum priming activity, can enhance plant defense responses against such challenges. In this work, we evaluated ARE as a potential priming agent in potato by analyzing pathogen-free tubers to identify cultivars with contrasting susceptibility based on constitutive metabolomic profiles and defense marker gene expression. Molecular signatures of constitutive resistance in Innovator This analysis revealed distinct cultivar-specific metabolic states, with Innovator consistently separating from susceptible backgrounds. Innovator leaves accumulated higher levels of trigonelline, linoleic acid-related signals, tryptophan-derived indolic metabolites, and several unassigned features, whereas susceptible cultivars such as Spunta showed elevated asparagine and other low molecular weight signals. Notably, the five unassigned signals (U_32_1, U_87_2, U_33_4, U_38_2, and U_115_1) may represent yet undescribed metabolites with potential as novel chemical indicators of basal resistance. The differential accumulation of these compounds, in parallel with increased transcript abundance of SA-responsive markers such as PR1 , support a constitutively primed metabolic state likely contributing to high resistance in Innovator. Among these features, trigonelline (N-methylnicotinate) emerges as a particularly interesting candidate. In potato, ¹H NMR-based metabolomics revealed trigonelline accumulation in SAMDC (S-adenosylmethionine decarboxylase) transgenic lines, with moderate levels linked to protective functions (Defernez et al. 2004 ). Its primary synthesis in green tissues, potentially linked to chloroplast metabolism, together with its capacity to be recycled into nicotinic acid for NAD biosynthesis, suggests additional roles in metabolite recycling, detoxification, and cellular regulation (Katahira and Ashihara 2009 ). Beyond its central metabolic role, trigonelline has emerged as a metabolite consistently linked to biotic defense. In tomato, its accumulation is triggered by Alternaria alternata infection or chitin treatment, where it contributes to pattern-triggered immunity by inhibiting fungal growth at physiologically relevant concentrations (Muñoz Hoyos et al. 2024 ). Similarly, recent NMR-based metabolomic analysis in tomato brown rugose fruit virus (ToBRFV)-infected plants showed strong trigonelline accumulation, further reinforcing its role as a defense-associated metabolite across plant species (Salmerón et al. 2025 ). Collectively, these findings position trigonelline at the interface of primary metabolism and inducible defense, with its constitutive accumulation in Innovator likely contributing to a primed resistance state. In parallel, basal enrichment of linoleic acid, a polyunsaturated fatty acid, underscores lipid remodeling as a complementary layer of this primed configuration and links jasmonate biosynthesis with broad stress adaptation (He and Ding 2020 ). This defense profile likely reinforces Innovator readiness to respond to environmental challenges. Complementing these results, tryptophan-derived 3-indolylmethyl metabolites, which are linked to glucosinolate pathways in Brassicaceae (Malcuit et al. 2000 ), were also elevated in Innovator, suggesting they may perform similar defense-related functions in Solanaceae. Their accumulation may involve uncharacterized pathways, potentially including glycosyltransferases such as UGT74B1, which was upregulated in Innovator, and may contribute to basal resistance. Consistently, the constitutive elevated levels of PR1 in Innovator support the involvement of SA-associated defense signaling in basal immunity, in line with previous reports linking basal PR expression to partial resistance, particularly through restricting P. infestans during the early biotrophic phase of infection (Vleeshouwers et al. 2000 ; Navarre and Mayo 2004 ; Halim et al. 2007 ). Supporting this, early untargeted liquid chromatography–mass spectrometry (LC-MS) metabolomics in the resistant cultivar Ziyun No.1 revealed elevated basal SA levels, phenylpropanoid intermediates, and triterpenoids, indicating metabolite-mediated defense priming (Zhu et al. 2021 ). In Innovator, a comparable primed metabolic state may accelerate defense responses and complement effector-triggered immunity for stronger pathogen restriction. Resistance architecture and infection outcomes The differences in immune status defense profile align with the resistance architecture described for these cultivars, as Innovator carries the Nb gene (PVX ROTH1) and Rpi-R1 , Rpi-R2 -like, Rpi-R3a and Rpi-R3b ( P. infestans ), whereas Spunta lacks functional resistance determinants. Results from the PVX ROTH1 and PiNSL-19 infection assays corroborated the observed defense profiles. Interestingly, Notably, while the PiNSL-19 genome is devoid of Avr1 , it encompasses several homologs of the Avr2 gene family (Jaurez 2024, Juarez et al. 2024 ), and the isolate was unable to overcome Innovator, indicating that at least one corresponding R–Avr interaction remains functional. By contrast, although Spunta cultivar carries Rpi-R1 (Armstrong et al. 2019 ), this resistance is ineffective against virulent isolates in which Avr1 alleles evade recognition, illustrating the limited durability of single-gene-resistance (Coomber et al. 2024 ). ARE-mediated defense priming in susceptible potato cultivars ARE treatment significantly reduces PVX ROTH1 accumulation in Spunta and in the susceptible cultivar Pentland Ivory (genotype nb ), correlating with increased transcription of SA-related defense genes ( PAL , ICS1 , PR1 ). These results align with previous findings in N. tabacum , where ARE mitigated viral symptoms via redox and hormonal modulation (Martin et al. 2023 ), and in S. lycopersicum , where RA treatment modulated redox signaling and delayed fruit ripening (Zhu et al. 2021 ). Notably, ARE also enhanced resistance in the PVX-ROTH1-resistant genotypes carrying Nb , suggesting additive effects with the effector-triggered immunity and highlighting its potential as a priming agent across different genetic backgrounds. The stronger induction of PR1 and PAL observed in Pentland Ivory is consistent with the central role of SA signaling in antiviral defense (Sánchez et al. 2010 ). In potato, endogenous SA levels tightly correlate with basal PR1 expression, a canonical marker of SA-dependent immune activation (Vleeshouwers et al. 2000 ; Navarre and Mayo 2004 ). Thus, the contrasting transcriptional responsiveness of Spunta and Pentland Ivory likely reflects intrinsic differences in SA pathway capacity, providing a mechanistic basis for the genotype-specific amplitude of ARE-mediated priming during PVX infection. Comparable genotype-dependent variability has been reported for β-aminobutyric acid (BABA), a well-established immune-priming compound. In tomato, BABA induces strong PR1 accumulation and enhances resistance to P. infestans (Cohen and Gisi 1994 ), nevertheless the magnitude and stability of this response vary considerably depending on plant genotype and on the controlled environmental conditions under which priming is induced (Cohen, 2002 ). Overall, the data indicate that genotype-dependent differences in SA responsiveness shape both the detectability of PR1 induction and the amplitude of ARE-mediated priming. In Spunta, ARE conferred moderate but reproducible protection against P. infestans , with lesion size reduced by approximately 30%. Notably, this physiological protection was not accompanied by significant changes in the expression of canonical SA- or JA-associated defense genes, consistent with the intrinsically limited SA responsiveness of this cultivar. In our experiments, a clear difference in PR1 induction between water- and ARE-treated plants was observed only at 48 hpi with PVX ROTH1, indicating a delayed transcriptional response consistent with the genotype-specific SA responsiveness of Spunta. Accordingly, ARE conferred measurable protection against both P. infestans and PVX even in the absence of robust early PR1 activation, suggesting a primed state operating through defense mechanisms not captured by classical hormonal markers, such as altered pathogen perception, reinforcement of physical barriers, or redox-based adjustments. Similar temporal constraints have been described for jasmonate-mediated priming, where early signaling events rapidly occur but transcriptional activation of defense genes is only detectable within a defined time window after stimulus (Arevalo-Marín et al. 2021), highlighting the importance of sampling timing. In summary, ARE primes potato defenses in a genotype-dependent manner, providing measurable protection against PVX and P. infestans even in the absence of early PR1 induction, and highlighting its potential for enhancing innate immunity also in this crop. In summary, ARE primes potato defenses in a genotype-dependent manner, providing measurable protection against PVX and P. infestans , and highlighting its potential for enhancing innate immunity in this crop. Conclusions and future perspectives Collectively, these findings demonstrate that priming with natural products, such as ARE, provides a functional bridge between constitutive and inducible resistance mechanisms, thereby complementing innate defense layers. The broad-spectrum effectiveness of ARE across diverse plant species, including Nicotiana , Glycine , Citrus , and Solanum , highlights its considerable versatility as a defense elicitor. Furthermore, the established metabolic and gene expression signatures of the resistant cultivar Innovator offer a robust reference framework for evaluating and optimizing future priming strategies. The observed genotype-dependent differences in SA responsiveness and PR1 inducibility suggest that ARE priming outcomes can be significantly optimized by selecting cultivars with favorable underlying metabolic and immune backgrounds. To further refine these strategies, future work should focus on integrating metabolite profiling with the targeted evaluation of ARE and synergistic compounds beyond RA. This will enable tailored approaches for both genetically uniform, susceptible cultivars like Spunta and more resistant backgrounds such as Innovator. Ultimately, field studies under variable climate and pathogen pressures will be essential to validate the laboratory findings and assess the practical benefits of ARE priming for effective disease management. References Agehara S, Pride L, Gallardo M, Hernandez-Monterroza J (2020) A simple, inexpensive, and portable image-based technique for nondestructive leaf area measurements: HS1395, 11/2020. EDIS 2020. https://doi.org/10.32473/EDIS-HS1395-2020 . Aremu AO, Omogbene TO, Fadiji T, Lawal IO, Opara UL, Fawole OA (2024) Plants as an alternative to the use of chemicals for crop protection against biotic threats: trends and future perspectives. Eur J Plant Pathol 170:711–766. https://doi.org/10.1007/s10658-024-02924-y Arévalo-Marín DF, Briceño-Robles DM, Mosquera T, Melgarejo LZ, Sarmiento F (2021). Jasmonic acid priming of potato uses hypersensitive response-dependent defense and delays necrotrophic phase change against Phytophthora infestans. Physiol Mol Plant Pathol 115: 101680. https://doi.org/10.1016/j.pmpp.2021.101680 Armstrong MR, Vossen J, Lim TY, et al (2019) Tracking disease resistance deployment in potato breeding by enrichment sequencing. Plant Biotechnol J 17:540–549. https://doi.org/10.1111/pbi.12997 Balint-Kurti P (2019) The plant hypersensitive response: concepts, control and consequences. Mol Plant Pathol 20:1163–1178. https://doi.org/10.1111/mpp.12821 Baulcombe DC, Chapman S, Santa Cruz S (1995) Jellyfish green fluorescent protein as a reporter for virus infections. Plant J 7:1045–1053. https://doi.org/10.1046/j.1365-313x.1995.07061045.x Bradshaw JE (2022) A brief history of the impact of potato genetics on the breeding of tetraploid potato cultivars for tuber propagation. Potato Res 65:461–501. https://doi.org/10.1007/s11540-021-09517-w Coban F, Ozer H, Yilmaz B, Lan Y (2025) Characterization of bioactive compounds in fenugreek genotypes in varying environments: diosgenin, trigonelline, and 4-hydroxyisoleucine. Front Plant Sci 16:1562931. https://doi.org/10.3389/fpls.2025.1562931 Cohen Y, Gisi U (1994) Systemic translocation of 14C-dl-3-aminobutyric acid in tomato plants in relation to induced resistance against Phytophthora infestans. Physiol Mol Plant Pathol 45:441–456. https://doi.org/10.1016/S0885-5765(05)80041-4 Cohen Y, Gisi U (2002) β-Aminobutyric Acid-induced resistance against plant pathogens. Plant Dis 86:448–457. https://doi.org/10.1094/PDIS.2002.86.5.448 Coomber A, Saville A, Ristaino JB (2024) Evolution of Phytophthora infestans on its potato host since the Irish potato famine. Nat Commun 15:1–12. https://doi.org/10.1038/s41467-024-50749-4 Defernez M, Gunning YM, Parr AJ, Shepherd LVT, Davies HV, Colquhoun IJ (2004) NMR and HPLC-UV profiling of potatoes with genetic modifications to metabolic pathways. J Agric Food Chem 52:6075–6085. https://doi.org/10.1021/jf049522e Dessì D, Fais G, Sarais G (2025) Nutritional and chemical characterization of red and purple potato peels: A polyphenol-rich by-product. Foods 14:1740. https://doi.org/10.3390/FOODS14101740 Devaux A, Goffart JP, Petsakos A, et al (2019) Global food security, contributions from sustainable potato agri-food systems. In: The potato crop: Its agricultural, nutritional and social contribution to humankind, Springer I. pp 3–35 Dieterle F, Ross A, Schlotterbeck G, Senn H (2006) Probabilistic quotient normalization as robust method to account for dilution of complex biological mixtures. Application in 1H NMR metabolomics. Anal Chem 78:4281–4290. https://doi.org/10.1021/ac051632c Favaro MA, Molina MC, Roeschlin RA, Gadea J, Gariglio N, Marano MR (2020) Different responses in mandarin cultivars uncover a role of cuticular waxes in the resistance to citrus canker. Phytopathology 110:1791–1801. https://doi.org/10.1094/PHYTO-02-20-0053-R Fry WE (2016) Phytophthora infestans: New Tools (and old ones) lead to new understanding and precision management. Annu Rev Phytopathol 54:529–547. https://doi.org/10.1146/annurev-phyto-080615-095951 Gabriel J, Coca A, Plata G, Parlevliet JE (2007) Characterization of the resistance to Phytophthora infestans in local potato cultivars in Bolivia. Euphytica 153:321–328. https://doi.org/10.1007/s10681-006-9237-x Garcia L, Gerhardt N, Martin AP, Martínez MF, Alemano S, Marano MR (2023) Tobacco necrosis virus A overcomes local cell death response in Nicotiana tabacum . Plant Pathol 72:154–169. https://doi.org/10.1111/ppa.13629 Garrett KA, Bebber DP, Etherton BA, et al (2022) Climate change effects on pathogen emergence: Artificial intelligence to translate big data for mitigation. Annu Rev Phytopathol 60:357–378. https://doi.org/10.1146/annurev-phyto-021021-042636 Ghislain M, Núñez J, Del Rosario HM, et al (2009) Robust and highly informative microsatellite-based genetic identity kit for potato. Mol Breed 23:377–388. https://doi.org/10.1007/S11032-008-9240-0 Greenberg JT, Yao N (2004) The role of regulation of programmed cell death in plant-pathogen interactions. Cell Microbiol 6:201–211. https://doi.org/10.1111/j.1462-5822.2004.00361.x Grubb CD, Zipp BJ, Ludwig-Müller J, Masuno MN, Molinski TF, Abel S (2004) Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis. Plant J 40:893–908. https://doi.org/10.1111/j.1365-313X.2004.02261.x Halim VA, Eschen-Lippold L, Altmann S, Birschwilks M, Scheel D, Rosahl S (2007) Salicylic acid is important for basal defense of Solanum tuberosum against Phytophthora infestans . Mol Plant-Microbe Interact 20:1346–1352. https://doi.org/10.1094/MPMI-20-11-1346 , He M, Ding NZ (2020) Plant unsaturated fatty acids: Multiple roles in stress response. Front Plant Sci 11:562785. https://doi.org/10.3389/fpls.2020.562785 Huang S, Vleeshouwers VGAA, Visser RGF, Jacobsen E (2005) An accurate in vitro assay for high-throughput disease testing of Phytophthora infestans in potato. Plant Dis 89:1263–1267. https://doi.org/10.1094/PD-89-1263 Juarez ME (2024) Caracterización poblacional, transcriptómica y análisis funcional de Phytophthora infestans en Argentina para la incorporación de resistencia al tizón tardío de la papa. Dissertation (PhD), Universidad de Buenos Aires. Juarez ME, Azcue J, Cano Mogrovejo ML, Lamour K, Bravo-Almonacid FF, Lucca AMF, Segretin ME (2024) Effector repertoire of an Argentinean Phytophthora infestans isolate and its relevance for late blight resistance deployment in potato. In: BSPP Plant Pathology 2024. Zenodo. https://doi.org/10.5281/zenodo.17087054 Katahira R, Ashihara H (2009) Profiles of the biosynthesis and metabolism of pyridine nucleotides in potatoes (Solanum tuberosum L.). Planta 231:35–45. https://doi.org/10.1007/s00425-009-1023-2 Kerchev P, van der Meer T, Sujeeth N, et al (2020) Molecular priming as an approach to induce tolerance against abiotic and oxidative stresses in crop plants. Biotechnol Adv 40: 107503. https://doi.org/10.1016/j.biotechadv.2019.107503 Kim HK, Choi YH, Verpoorte R (2010) NMR-based metabolomic analysis of plants. Nat Protoc 5:536–549. https://doi.org/10.1038/nprot.2009.237 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-∆∆CT method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262 Malcuit I, De Jong W, Baulcombe DC, Shields DC, Kavanagh TA (2000) Acquisition of multiple virulence/avirulence determinants by potato virus X (PVX) has occurred through convergent evolution rather than through recombination. Virus Genes 20:165–172. https://doi.org/10.1023/A:1008178800366 Malcuit I, Marano MR, Kavanagh TA, De Jong W, Forsyth A, Baulcombe DC (1999) The 25-kDa movement protein of PVX elicits Nb-mediated hypersensitive cell death in potato. Mol Plant-Microbe Interact 12:536–543. https://doi.org/10.1094/MPMI.1999.12.6.536 Marano MR, Malcuit I, De Jong W, Baulcombe DC (2002) High-resolution genetic map of Nb, a gene that confers hypersensitive resistance to potato virus X in Solanum tuberosum. Theor Appl Genet 105:192–200. https://doi.org/10.1007/s00122-002-0962-9 Martin AP, Martínez MF, Chiesa MA, et al (2023) Priming crop plants with rosemary ( Salvia rosmarinus Spenn, syn Rosmarinus officinalis L.) extract triggers protective defense response against pathogens. Plant Physiol Biochem 197: 107644. https://doi.org/10.1016/j.plaphy.2023.107644 Mondino MC, Grasso R, Balaban D, Ortiz MM (2021) Censo 2021 del Cinturón hortícola de Rosario. https://www.argentina.gob.ar/sites/default/files/2025/07/inta_oliveros_censo_horticola_2021.pdf . Accessed 18 December 2025. Muñoz Hoyos L, Anisha WP, Meng C, et al (2024) Untargeted metabolomics reveals PTI-associated metabolites. Plant Cell Environ 47:1224–1237. https://doi.org/10.1111/pce.14794 Navarre DA, Brown CR, Sathuvalli VR (2019) Potato vitamins, minerals and phytonutrients from a plant biology perspective. Am J Potato Res 96:111–126. https://doi.org/10.1007/s12230-018-09703-6 Navarre DA, Mayo D (2004) Differential characteristics of salicylic acid-mediated signaling in potato. Physiol Mol Plant Pathol 64:179–188. https://doi.org/10.1016/j.pmpp.2004.09.001 Nicaise V (2014) Crop immunity against viruses: Outcomes and future challenges. Front Plant Sci 5:118474. https://doi.org/10.3389/FPLS.2014.00660/XML Nyalugwe EP, Wilson CR, Coutts BA, Jones RAC (2012) Biological properties of potato virus x in potato: Effects of mixed infection with potato virus s and resistance phenotypes in cultivars from three continents. Plant Dis 96:43–54. https://doi.org/10.1094/PDIS-04-11-0305 Osbourn AE (1996) Preformed antimicrobial compounds and plant defense against fungal attack. Plant Cell 8:1821–1831. https://doi.org/10.1105/tpc.8.10.1821 Pacheco R, García-Marcos A, Barajas D, Martiáñez J, Tenllado F (2012) PVX-potyvirus synergistic infections differentially alter microRNA accumulation in Nicotiana benthamiana . Virus Res 165:231–235. https://doi.org/10.1016/j.virusres.2012.02.012 Pang Z, Lu Y, Zhou G, et al (2024) MetaboAnalyst 6.0: towards a unified platform for metabolomics data processing, analysis and interpretation. Nucleic Acids Res 52:398–406. https://doi.org/10.1093/nar/gkae253 PW T (1988) Use of uncontrolled freezing for liquid nitrogen storage of Phytophthora species. Plant Dis 72:680–682 Répás Z, Nagy R, Polgár ZG, Győri Z (2024) Study to determine the nutritional characteristics of potato varieties that are suitable for the application of the freeze-drying process. Potato Res 1–23. https://doi.org/10.1007/s11540-024-09827-9 Salam U, Ullah S, Tang ZH, et al (2023) Plant metabolomics: An overview of the role of primary and secondary metabolites against different environmental stress factors. Life (Basel) 13:706. https://doi.org/10.3390/LIFE13030706 Salmerón A del M, Abreu AC, Tristán AI, et al (2025) Metabolic profiling of tomato plants infected with tomato brown rugose fruit virus: Insights into plant defense mechanisms and potential prebiotic interventions. ACS Agric Sci Technol 5:714–724. https://doi.org/10.1021/acsagscitech.4c00557 Sambrook, J.; Fritsch, E.F.; Maniatis T (1989) Molecular Cloning: A laboratory manual, 2nd edn. Cold Spring Harbor, NY, U.S.A. Sánchez G, Gerhardt N, Siciliano F, Vojnov A, Malcuit I, Marano MR (2010) 394–405 Salicylic acid is involved in the Nb-mediated defense responses to potato virus X Solanum tuberosum . Mol Plant-Microbe Interact 23:394–405. https://doi.org/10.1094/MPMI-23-4-0394 Savary S, Willocquet L, Pethybridge SJ, Esker P, McRoberts N, Nelson A (2019) The global burden of pathogens and pests on major food crops. Nat Ecol Evol 3:430–439. https://doi.org/10.1038/s41559-018-0793-y Syller J, Grupa A (2016) Antagonistic within-host interactions between plant viruses: Molecular basis and impact on viral and host fitness. Mol Plant Pathol 17:769–782. https://doi.org/10.1111/MPP.12322 Tagliotti ME, Deperi SI, Bedogni MC, et al (2018) Use of easy measurable phenotypic traits as a complementary approach to evaluate the population structure and diversity in a high heterozygous panel of tetraploid clones and cultivars. BMC Genet 19:1–12. https://doi.org/10.1186/s12863-017-0556-9 Tiwari JK, Buckseth T, Challam C, et al (2022a) CRISPR/Cas genome editing in potato: Current status and future perspectives. Front Genet 13:1–6. https://doi.org/10.3389/fgene.2022.827808 Tiwari JK, Buckseth T, Zinta R, et al (2022b) Germplasm, breeding, and genomics in potato improvement of biotic and abiotic stresses tolerance. Front Plant Sci 13: 805671. https://doi.org/10.3389/fpls.2022.805671 van Wersch S, Tian L, Hoy R, Li X (2020) Plant NLRs: The whistleblowers of plant immunity. Plant commun.1:100016. https://doi.org/10.1016/j.xplc.2019.100016 Van West P, De Jong AJ, Judelson HS, Emons, Anne MC, Govers F (1998) The ipiO gene of Phytophthora infestans is highly expressed in invading hyphae during infection. Fungal Genet Biol 23:126–138. https://doi.org/10.1006/fgbi.1998.1036 Verchot J (2022) Potato virus X: A global potato-infecting virus and type member of the Potexvirus genus. Mol Plant Pathol 23:315–320. https://doi.org/10.1111/mpp.13163 Veselkov KA, Lindon JC, Ebbels TMD, et al (2009) Recursive segment-wise peak alignment of biological 1H NMR spectra for improved metabolic biomarker recovery. Anal Chem 81:56–66. https://doi.org/10.1021/ac8011544 Vleeshouwers VGAA, Van Dooijeweert W, Govers F, Kamoun S, Colon LT (2000) Does basal PR gene expression in Solanum species contribute to non-specific resistance to Phytophthora infestans ? Physiol Mol Plant Pathol 57:35–42. https://doi.org/10.1006/pmpp.2000.0278 Vollmer R, Espirilla J, Villagaray R, et al (2021) Cryopreservation of potato shoot tips for long-term storage. In: Methods in molecular biology. Humana Press Inc., pp 21–54 Wishart DS, Jewison T, Guo AC, et al (2013) HMDB 3.0-The human metabolome database in 2013. Nucleic Acids Res 41: D801-807. https://doi.org/10.1093/nar/gks1065 Wu EJ, Wang YP, Yang LN, Zhao MZ, Zhan J (2022) Elevating air temperature may enhance future epidemic risk of the plant pathogen Phytophthora infestans . J fungi (Basel) 8:808. https://doi.org/10.3390/JOF8080808 Yu XQ, Niu HQ, Liu C, Wang HL, Yin W, Xia X (2024) PTI-ETI synergistic signal mechanisms in plant immunity. Plant biotechnol J 22:2113–2128. https://doi.org/10.1111/pbi.14332 Zhang P, Jackson E, Li X, Zhang Y (2025) Salicylic acid and jasmonic acid in plant immunity. Hortic res 12:uhaf082. https://doi.org/10.1093/hr/uhaf082 Zhang S, Huang A, Lv X, et al (2023) Anti-oomycete effect and mechanism of salicylic acid on Phytophthora infestans. J Agric Food Chem 71:20613–20624. https://doi.org/10.1021/acs.jafc.3c05748 Zhu C, Wu S, Sun T, et al (2021) Rosmarinic acid delays tomato fruit ripening by regulating ripening-associated traits. Antioxidants 10:1821. https://doi.org/10.3390/antiox10111821 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.zip Electronic Supplementary material Table S1. Parentage and reported resistance genes to potato virus X (PVX) strain ROTH1 and Phytophthora infestans in the potato cultivars. Table S2. Optimized spectral data matrix derived from MCR-ALS integrated components for multivariate statistical analysis. Table S3. Gene name, primer sequences and quantitative PCR conditions. Fig. S1. Construction and expression of PVX ROTH1-GFP in Nicotiana benthamiana . a. Schematic representation of the plasmids used in this study. Except for the viral cDNA regions, the diagrams are schematic and not drawn to scale. Each construct contains sequences encoding the viral RNA-dependent RNA polymerase (RdRp), the movement proteins (25, 12, and 8 kDa) and the coat protein (CP). The CaMV 35S promoter (35S), the nopaline synthase (NOS) terminator, and the restriction sites used to generate the PVX ROTH1-GFPconstruct are shown in the diagram. bLeaves of Nicotiana benthamiana inoculated with the PVX ROTH1-GFP plasmid, 10 days post-inoculation (dpi), shown under white light and ultraviolet (UV) to visualize GFP fluorescence. Fig. S2. Experimental workflow for rosemary extract treatments and pathogen challenge. Foliar applications of aqueous rosemary extract (ARE) were performed by spraying every 24 h for 2 consecutive days. Forty-eight hours after the first treatment, plants were inoculated with pathogens. Symptom development was monitored to assess the protective effect of the extracts. Fig. S3. Differential metabolite accumulation of resistant and susceptible potato cultivars. a. Violin plots showing the normalized concentration for the annotated metabolites identified as significantly different between resistant and susceptible potato cultivars by the volcano plot analysis. b. Analysis of defense-related genes in potato leaves of different cultivars. Reverse transcription quantitative PCR (qPCR) of SA biosynthesis genes ( ICS1 , PAL1 ) and defense-related genes ( PR1 , SA signaling; UGT74B1 , glucosinolate metabolism) in Innovator (I) and Spunta (Sp). Values were normalized to the constitutive EF-α gene and fold-changes were expressed relative to Spunta. Means ± standard deviation of eight to ten replicates, each consisting of five leaves from ten different plants are shown. Asterisks indicate significant differences at p < 0.05 (one- way analysis of variance, Tukey's test). EF-α , ELONGATION FACTOR 1-α ; PAL , PHENYLALANINE AMMONIA-LYASE ; ICS , ISOCHORISMATE SYNTHASE ; PR1 , PATHOGENESIS-RELATED PROTEIN 1; UGT74B1 , UDP-GLUCOSYLTRANSFERASE 74B1 . Fig. S4. Nb-mediated hypersensitive response (HR) to the PVX ROTH1 effector 25K1 in potato cultivars. a. Potato cultivars Pentland Ivory (resistant, Nb ; susceptible, nb ), Innovator (I), Frital (F), Kennebec (K), and Spunta (Sp) were agroinfiltrated with either pBIN25K1 (black dashed lines) or pBIN25K3 (red dashed lines). Tissue samples were harvested at the 48 h post-infiltration. White arrows indicate hypersensitive response (HR). b. Analysis of genomic DNA isolated from leaves of potato cultivars Pentland Ivory ( nb and Nb ), Frital (F) and Innovator (I) was performed using markers SPUD237 and SPUD839. PCR products were digested with Alu I (SPUD237) or Alw N1 (SPUD839). DNA fragments linked to the Nb locus are indicated by an arrow. Fig. S5. Rosemary extract primes potato leaves against P. infestans . a. Leaves from 4–5-week-old Spunta plants were sprayed with aqueous rosemary extract (ARE) or water on two consecutive days, detached, and inoculated with P. infestans zoospores. Leaves were maintained in humid chambers, and lesion area proportion (lesioned area/total leaf area) was measured at 2-, 3- and 4-days post-inoculation (dpi) using ImageJ 1.54p. b.Box plots show median (line), interquartile range (box), and minimum and maximum values (whiskers). Fifteen samples were included in each assay. Statistical significance was determined by one-way analysis of variance (Sidak's test) ** p = 0.0003; **** p < 0.0001 . This experiment was repeated four times. Cite Share Download PDF Status: Published Journal Publication published 28 Mar, 2026 Read the published version in Plant Cell Reports → Version 1 posted Editorial decision: Revision requested 17 Jan, 2026 Reviews received at journal 14 Jan, 2026 Reviews received at journal 13 Jan, 2026 Reviewers agreed at journal 07 Jan, 2026 Reviewers agreed at journal 07 Jan, 2026 Reviewers agreed at journal 07 Jan, 2026 Reviewers invited by journal 07 Jan, 2026 Editor assigned by journal 20 Dec, 2025 Submission checks completed at journal 20 Dec, 2025 First submitted to journal 18 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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07:21:38","extension":"html","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":219932,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8399039/v1/23f7b183958557a267d88a17.html"},{"id":100358954,"identity":"7a2712f1-e861-4ab7-946f-d9fdab591e80","added_by":"auto","created_at":"2026-01-16 07:21:36","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":996510,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolic profiles in leaf of potato cultivars. a.\u003c/strong\u003e Principal component analysis (PCA) score plots, where each point represents one biological replicate (n = 5 per cultivar). Confidence ellipses illustrate clustering within cultivars (I, Innovator, K, Kennebec, F, Frital, Sp, Spunta). \u003cstrong\u003eb.\u003c/strong\u003e Orthogonal partial least squares-discriminant analysis (OPLS-DA) of metabolic fingerprints in potato cultivars. Cultivars were grouped as R (resistance-like, green) and S (susceptibility-like, red) based on the presence of resistance loci (\u003cem\u003eRpi-R\u003c/em\u003e, \u003cem\u003eNb\u003c/em\u003e). \u003cstrong\u003ec.\u003c/strong\u003eHeatmap of discriminant metabolites selected from multivariate analysis, depicting relative abundance patterns across cultivars. Hierarchical clustering was applied to both metabolites and samples. Color intensity reflects normalized metabolite levels. \u003cstrong\u003ed.\u003c/strong\u003e Volcano plot of differentially accumulated metabolites between R and S groups, defined by fold change (FC) \u0026gt; 2.0 (x axis) and a t-tests threshold adjusted of 0.01 (y axis), highlighting statistically significant metabolic differences under basal conditions. Red and blue dots indicate significant metabolites, and the gray dots below the FC threshold line represent statistically non-significant metabolites. Vertical FC threshold lines indicate increases or decreases in metabolites abundance in resistant-like compared to susceptibility-like cultivars.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8399039/v1/f4161a230c0bd6001b20b325.jpg"},{"id":100358681,"identity":"39da73a3-575b-414f-97f8-4b2a993858ce","added_by":"auto","created_at":"2026-01-16 07:21:14","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2052916,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential responses of potato cultivars to PVX ROTH1-GFP infection.\u003c/strong\u003e \u003cstrong\u003ea.\u003c/strong\u003e Progression of PVX ROTH1-GFP infection in\u003cem\u003e \u003c/em\u003epotato cultivars visualized under white light and laser scanning confocal microscopy. GFP fluorescence indicates viral replication and cell-to-cell movement in susceptible cultivars, whereas resistant cultivars display a hypersensitive response (HR, black dashed lines). Images were taken at 2 days post-inoculation (dpi) in resistant and 4 dpi in susceptible cultivars. \u003cstrong\u003eb\u003c/strong\u003e. Reverse transcription and quantitative PCR (qPCR) analysis of PVX ROTH1 coat protein (\u003cem\u003eCP\u003c/em\u003e) transcript accumulation in inoculated leaves of different potato cultivars. Fold changes of \u003cem\u003eCP\u003c/em\u003e RNA levels, normalized to EF-α, are relative to inoculated leaves at 48 h post-inoculation (hpi) in \u003cem\u003eNb\u003c/em\u003e genotype. Values are expressed as means ± standard deviation of eight samples. Different letters indicate significant differences at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (one- way analysis of variance, Tukey's test). \u003cem\u003eEF- α, Elongation Factor 1- α\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8399039/v1/5133643b72ea940b37bf8810.jpg"},{"id":100358609,"identity":"4fd575ee-48c7-458c-b746-2eb8e641c492","added_by":"auto","created_at":"2026-01-16 07:21:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":710935,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential responses of potato cultivars to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePhytophthora infestans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection.\u003c/strong\u003e \u003cstrong\u003ea.\u003c/strong\u003e \u003cem\u003eIn vitro\u003c/em\u003e-grown plantlets of Innovator, Frital, Kennebec and Spunta were inoculated with \u003cem\u003eP. infestans \u003c/em\u003eisolated\u003cem\u003e \u003c/em\u003ePiNSL-19. \u003cstrong\u003eb\u003c/strong\u003e. Detached leaf assays were performed using potato leaves placed on water-statured floral foam and inoculated with PiNSL-19. Lower panels show magnified views of lesion areas. Lesion area proportion (lesioned area/total leaf area) was quantified using ImageJ 1.54p at 4 days post-inoculation (dpi) and represented as box plots. Fifteen samples were included in each assay. Box plots indicate the median (line), interquartile range (box), and minimum and maximum values (whiskers). Different letters indicate statistically significant differences at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (one- way analysis of variance, Tukey's test). I, Innovator; K, Kennebec; F, Frital; Sp, Spunta.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8399039/v1/9a417060e63159f67c87a7c8.jpg"},{"id":99893342,"identity":"802d7070-4ca9-46dd-9de3-84c3ab0d48cf","added_by":"auto","created_at":"2026-01-09 14:14:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1407178,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAqueous rosemary extract (ARE) confers protection against PVX ROTH1-GFP in potato cultivars. a.\u003c/strong\u003e Potato leaves of cultivars Pentland Ivory (\u003cem\u003eNb\u003c/em\u003e and\u003cem\u003e nb\u003c/em\u003e) and Spunta (Sp) were treated with aqueous rosemary extract (ARE) or water, and then inoculated with PVX ROTH1-GFP. Leaves were imaged by laser scanning confocal microscopy at 2 days post-inoculation (dpi) in resistant and 4 dpi in susceptible cultivars. \u003cstrong\u003eb.\u003c/strong\u003e Reverse transcription and quantitative PCR (qPCR) analysis at 24 and 48 h post-inoculation (hpi) in PVX ROTH1-GFP-inoculated plants treated with ARE or water. Values were normalized to the constitutive \u003cem\u003eEF-α\u003c/em\u003egene and fold changes were expressed relative to inoculated water-treated leaves (controls) of Pentland Ivory \u003cem\u003enb\u003c/em\u003e genotype (\u003cem\u003enb\u003c/em\u003e) for each time. Mean ± standard deviation (n = 4 replicates, each consisting of four leaves from four different plants) are shown. Different letters indicate significant differences at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (one-way ANOVA, Tukey’s test). \u003cstrong\u003ec.\u003c/strong\u003e qPCR analysis of \u003cem\u003ePAL\u003c/em\u003e, \u003cem\u003eIC\u003c/em\u003e1, and \u003cem\u003ePR1\u003c/em\u003e at 24 and 48 hpi with PVX ROTH1-GFP in potato cultivars treated with ARE or water. Values were normalized to the constitutive \u003cem\u003eEF-α \u003c/em\u003egene and fold changes were expressed relative to inoculated water-treated leaves (control) at each time. Mean ± standard deviation of 5 replicates, each consisting of five leaves from five different plants are shown. Asterisks indicate significant differences at *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e= 0.008 (one-way ANOVA, Tukey’s test). \u003cem\u003eEF-α\u003c/em\u003e, \u003cem\u003eELONGATION FACTOR 1-α\u003c/em\u003e; \u003cem\u003ePAL\u003c/em\u003e, \u003cem\u003ePHENYLALANINE AMMONIA-LYASE\u003c/em\u003e;\u003cem\u003e ICS\u003c/em\u003e, \u003cem\u003eISOCHORISMATE SYNTHASE\u003c/em\u003e; \u003cem\u003ePR1\u003c/em\u003e, \u003cem\u003ePATHOGENESIS-RELATED PROTEIN 1\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8399039/v1/dd07375447d20d574bbb80e7.jpg"},{"id":99893345,"identity":"7959f8fd-f0ee-4a4a-9f6f-32215620f065","added_by":"auto","created_at":"2026-01-09 14:14:51","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":830619,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDefense responses of potato cv. Spunta to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePhytophthora infestans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection after treatment with aqueous rosemary extract (ARE) or water. a.\u003c/strong\u003e Representative leaves of cultivar Spunta treated with ARE or water prior to inoculation with \u003cem\u003eP. infestans \u003c/em\u003eisolate PiNSL-19, showing disease symptoms. \u003cstrong\u003eb.\u003c/strong\u003e Lesion area proportion (lesioned area/total leaf area) corresponding to the leaves in (\u003cstrong\u003ea\u003c/strong\u003e), represented as box plots from three independent experiments. Seven to ten samples were included in each assay. Box plots indicate the median (line), interquartile range (box), and minimum and maximum values (whiskers). \u003cstrong\u003ec.\u003c/strong\u003e Reverse transcription and quantitative PCR (qPCR) analysis at 48 and 72 h post-inoculation (hpi) in PiNSL-19-inoculated plants treated with ARE or water. Values were normalized to the constitutive \u003cem\u003eEF-α \u003c/em\u003egene and fold-changes were expressed relative to inoculated water-treated leaves (control) at 48 hpi. Means ± standard deviation of four replicates, each consisting of four leaves from four different plants are shown. Different letters indicate significant differences at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (one-way ANOVA, Tukey’s test). \u003cem\u003eEF-α\u003c/em\u003e, \u003cem\u003eELONGATION FACTOR 1-α\u003c/em\u003e; \u003cem\u003ePAL\u003c/em\u003e, \u003cem\u003ePHENYLALANINE AMMONIA-LYASE\u003c/em\u003e;\u003cem\u003e ICS\u003c/em\u003e, \u003cem\u003eISOCHORISMATE SYNTHASE\u003c/em\u003e; \u003cem\u003ePR1\u003c/em\u003e, \u003cem\u003ePATHOGENESIS-RELATED PROTEIN 1\u003c/em\u003e; \u003cem\u003eACX1/ACX2\u003c/em\u003e, \u003cem\u003eACYL-COA OXIDASE 1/2\u003c/em\u003e; AOS, \u003cem\u003eALLENE OXIDE SYNTHASE 2\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8399039/v1/71095fe91be0c0647f366394.jpg"},{"id":105755464,"identity":"2943c263-5abe-40e2-8a5f-a3c3e1390811","added_by":"auto","created_at":"2026-03-30 16:27:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8390680,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8399039/v1/a05b084d-3a9d-4416-beba-7e53e6c1bf16.pdf"},{"id":100358987,"identity":"ac96c602-e785-47cd-b2d3-5771fb825f70","added_by":"auto","created_at":"2026-01-16 07:21:36","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2675370,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElectronic Supplementary material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S1. Parentage and reported resistance genes to potato virus X (PVX) strain ROTH1 and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePhytophthora infestans \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ein the potato cultivars.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S2. Optimized spectral data matrix derived from MCR-ALS integrated components for multivariate statistical analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S3. Gene name, primer sequences and quantitative PCR conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S1. Construction and expression of PVX ROTH1-GFP in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNicotiana benthamiana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ea.\u003c/strong\u003e Schematic representation of the plasmids used in this study. Except for the viral cDNA regions, the diagrams are schematic and not drawn to scale. Each construct contains sequences encoding the viral RNA-dependent RNA polymerase (RdRp), the movement proteins (25, 12, and 8 kDa) and the coat protein (CP). The CaMV 35S promoter (35S), the nopaline synthase (NOS) terminator, and the restriction sites used to generate the PVX ROTH1-GFPconstruct are shown in the diagram. \u003cstrong\u003eb\u003c/strong\u003eLeaves of \u003cem\u003eNicotiana benthamiana\u003c/em\u003e inoculated with the PVX ROTH1-GFP plasmid, 10 days post-inoculation (dpi), shown under white light and ultraviolet (UV) to visualize GFP fluorescence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S2. Experimental workflow for rosemary extract treatments and pathogen challenge. \u003c/strong\u003eFoliar applications of aqueous rosemary extract (ARE) were performed by spraying every 24 h for 2 consecutive days. Forty-eight hours after the first treatment, plants were inoculated with pathogens. Symptom development was monitored to assess the protective effect of the extracts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S3. Differential metabolite accumulation of resistant and susceptible potato cultivars. a.\u003c/strong\u003e Violin plots showing the normalized concentration for the annotated metabolites identified as significantly different between resistant and susceptible potato cultivars by the volcano plot analysis. \u003cstrong\u003eb.\u003c/strong\u003e Analysis of defense-related genes in potato leaves of different cultivars. Reverse transcription quantitative PCR (qPCR) of SA biosynthesis genes (\u003cem\u003eICS1\u003c/em\u003e, \u003cem\u003ePAL1\u003c/em\u003e) and defense-related genes (\u003cem\u003ePR1\u003c/em\u003e, SA signaling; \u003cem\u003eUGT74B1\u003c/em\u003e, glucosinolate metabolism) in Innovator (I) and Spunta (Sp). Values were normalized to the constitutive \u003cem\u003eEF-α \u003c/em\u003egene and fold-changes were expressed relative to Spunta. Means ± standard deviation of eight to ten replicates, each consisting of five leaves from ten different plants are shown. Asterisks indicate significant differences at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (one- way analysis of variance, Tukey's test). \u003cem\u003eEF-α\u003c/em\u003e, \u003cem\u003eELONGATION FACTOR 1-α\u003c/em\u003e; \u003cem\u003ePAL\u003c/em\u003e, \u003cem\u003ePHENYLALANINE AMMONIA-LYASE\u003c/em\u003e;\u003cem\u003e ICS\u003c/em\u003e, \u003cem\u003eISOCHORISMATE SYNTHASE\u003c/em\u003e; \u003cem\u003ePR1\u003c/em\u003e, \u003cem\u003ePATHOGENESIS-RELATED PROTEIN 1; UGT74B1\u003c/em\u003e,\u003cem\u003e UDP-GLUCOSYLTRANSFERASE 74B1\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S4. Nb-mediated hypersensitive response (HR) to the PVX ROTH1 effector 25K1 in potato cultivars.\u003c/strong\u003e \u003cstrong\u003ea.\u003c/strong\u003e Potato cultivars Pentland Ivory (resistant, \u003cem\u003eNb\u003c/em\u003e; susceptible, \u003cem\u003enb\u003c/em\u003e), Innovator\u003cem\u003e \u003c/em\u003e(I), Frital (F), Kennebec\u003cem\u003e \u003c/em\u003e(K), and Spunta (Sp) were agroinfiltrated with either pBIN25K1 (black dashed lines) or pBIN25K3 (red dashed lines). Tissue samples were harvested at the 48 h post-infiltration. White arrows indicate hypersensitive response (HR). \u003cstrong\u003eb.\u003c/strong\u003e Analysis of genomic DNA isolated from leaves of potato cultivars Pentland Ivory (\u003cem\u003enb\u003c/em\u003e and \u003cem\u003eNb\u003c/em\u003e), Frital (F) and Innovator (I) was performed using markers SPUD237 and SPUD839. PCR products were digested with \u003cem\u003eAlu\u003c/em\u003eI (SPUD237) or \u003cem\u003eAlw\u003c/em\u003eN1 (SPUD839). DNA fragments linked to the \u003cem\u003eNb\u003c/em\u003e locus are indicated by an arrow.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S5. Rosemary extract primes potato leaves against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. infestans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. a. \u003c/strong\u003eLeaves from 4–5-week-old Spunta plants were sprayed with aqueous rosemary extract (ARE) or water on two consecutive days, detached, and inoculated with \u003cem\u003eP. infestans\u003c/em\u003e zoospores. Leaves were maintained in humid chambers, and lesion area proportion (lesioned area/total leaf area) was measured at 2-, 3- and 4-days post-inoculation (dpi) using ImageJ 1.54p. \u003cstrong\u003eb.\u003c/strong\u003eBox plots show median (line), interquartile range (box), and minimum and maximum values (whiskers). Fifteen samples were included in each assay. Statistical significance was determined by one-way analysis of variance (Sidak's test) ** \u003cem\u003ep = \u003c/em\u003e0.0003; **** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001\u003cem\u003e. \u003c/em\u003eThis experiment was repeated four times.\u003c/p\u003e","description":"","filename":"Supplementarymaterial.zip","url":"https://assets-eu.researchsquare.com/files/rs-8399039/v1/35128b0ad8a13c2bea1a08d8.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Rosemary extract primes cultivar-dependent defense responses in potato against pathogen attack","fulltext":[{"header":"Key message","content":"\u003cp\u003eConstitutive defense profiles differentiate potato resistance to PVX and . Aqueous rosemary extract (ARE) primes susceptible cultivars, offering a sustainable strategy to boost resilience against these major foliar pathogens.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003ePotato (\u003cem\u003eSolanum tuberosum\u003c/em\u003e L.) plays a crucial role in global agriculture due to its phenotypic plasticity and adaptability to diverse environments. With over 4,000 varieties, primarily originating from the Andes, and an annual production exceeding 350\u0026nbsp;million tons, potatoes rank among the top crops alongside maize, wheat, and rice (Devaux et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Supporting the subsistence of over 1\u0026nbsp;billion people, potatoes provide a vital source of carbohydrates, proteins, dietary fibers and micronutrients (Navarre et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003ep\u0026aacute;s et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Their skin contributes essential vitamins and minerals that enhance the overall nutritional value (Dess\u0026igrave; et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMost commercial potatoes belong to autotetraploid cultivars of \u003cem\u003eSolanum tuberosum\u003c/em\u003e subsp. \u003cem\u003etuberosum\u003c/em\u003e L, which displays remarkable diversity in traits such as phenological maturity, yield, and tuber characteristics including shape, size, and skin color (Tagliotti et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Bradshaw \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Their versatility is further highlighted by the wide range of phytochemical profiles and the existence of market classes adapted for fresh consumption, processing, or seed production (Navarre et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Despite advances in breeding and genomics, potato production continues to face major challenges, including adverse environmental conditions, climate change, and pathogen pressures (Tiwari et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003ea\u003c/span\u003e). Climate-driven changes in temperature, humidity, and precipitation are reshaping plant\u0026ndash;pathogen interactions worldwide, influencing pathogen distribution, virulence, and epidemic dynamics (Garrett et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Among the most devastating pathogens, \u003cem\u003ePhytophthora infestans\u003c/em\u003e (Mont.) de Bary, the causal agent of late blight, has shown remarkable adaptability not only to fluctuating temperatures but also to fungicide pressure, resulting in extended epidemic potential under warming scenarios (Wu et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This oomycete remains a major constraint for potato production globally, capable of causing yield losses of 70\u0026ndash;80% under conducive environmental conditions (Fry \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Savary et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Viral diseases such as Potato virus X (PVX) substantially contribute to global potato yield losses, particularly under co-infections with potyviruses and luteoviruses, where reductions of up to 80% have been reported (Pacheco et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Nicaise \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Syller and Grupa \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Verchot \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Although resistance to PVX and \u003cem\u003eP. infestans\u003c/em\u003e has been previously characterized in commercial cultivars under field conditions, it remains unclear whether these resistance traits persist under current environmental and agronomic pressures. Therefore, developing effective and sustainable disease management strategies to maintain crop resilience and productivity remain major challenges. In this framework, natural plant extracts have emerged as promising bioprotectants against pathogens, capable of triggering defense priming and enhancing immunity without the metabolic cost of constitutive resistance (Kerchev et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Aremu et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). For instance, rosemary (\u003cem\u003eSalvia rosmarinus\u003c/em\u003e Spenn) aqueous extracts (ARE), rich in phenolics such as rosmarinic acid (RA), have been shown to induce plant defense responses against a broad range of pathogens, including virus, bacteria, and fungi (Martin et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePlant immunity relies on a multilayered defense system integrating constitutive and inducible responses. Preformed antimicrobial metabolites and structural barriers provide basal protection (Osbourn \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Vleeshouwers et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Navarre and Mayo \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Halim et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Favaro et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Upon pathogen perception, pattern-recognition receptors (PRRs) and intracellular resistance (R) proteins, most of which belong to the nucleotide-binding leucine-rich repeat (NLR) family (van Wersch et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Coban et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), activate defense signaling cascades that culminate in a hypersensitive response (HR) characterized by programmed cell death that restricts pathogen spread (Greenberg and Yao \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Balint-Kurti \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we analyzed the constitutive defense responses of four commercially potato cultivars -Innovator, Kennebec, Spunta and Frital-INTA - grown in major potato-producing regions of Argentina (Mondino, M.C.; Grasso, R.; Balaban, D.; Ortiz Mackinson, M.; Cardozo, F.; Timoni, R.; Vita Larrieu 2021). Metabolomic and molecular analyses performed on non-inoculated leaves revealed distinct basal defense profiles among cultivars. Innovator showed higher accumulation of defense-related metabolites and stronger expression of defense-associated genes, consistent with its resistance phenotype, whereas Spunta exhibited a weak basal defense signature indicative of high susceptibility to both pathogens (Nyalugwe et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Armstrong et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Controlled inoculation assays with \u003cem\u003ePotato virus X\u003c/em\u003e (PVX) and \u003cem\u003eP. infestans\u003c/em\u003e confirmed these contrasting responses. Based on these results, Spunta was selected as a model to evaluate priming strategies aimed at enhancing defense activation under field-relevant conditions. ARE, applied prior to pathogen inoculation, significantly reduced PVX accumulation and late blight lesion severity, reinforcing defense responses through a priming mechanism in this susceptible genotype.\u003c/p\u003e \u003cp\u003eOur findings demonstrate that cultivar-specific metabolomic and transcriptional signatures underlie resistance to PVX and \u003cem\u003eP. infestans\u003c/em\u003e, and that ARE-mediated priming effectively enhances potato immunity. This approach represents a sustainable strategy to strengthen crop resilience against foliar pathogens under changing climatic conditions.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGermplasm and clonal propagation of potato cultivars\u003c/h2\u003e \u003cp\u003eTubers of \u003cem\u003eSolanum tuberosum\u003c/em\u003e L. cultivars Spunta, Kennebec, Innovator and Frital-INTA (hereafter referred to as Frital) were obtained from potato growers across the main horticultural area of Rosario, Argentina, extending roughly between 33\u0026deg;S and 60\u0026deg;W (Mondino, M.C.; Grasso, R.; Balaban, D.; Ortiz Mackinson, M.; Cardozo, F.; Timoni, R.; Vita Larrieu 2021).\u003c/p\u003e \u003cp\u003eCultivar identity was confirmed by specialists from National Agricultural Technology Institute (INTA), using molecular markers (Ghislain et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The cultivars used in this study are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, together with information on their parentage and previously reported resistance (\u003cem\u003eR\u003c/em\u003e) genes associated with the pathogens analyzed herein. Tubers harvested from mature plants were stored at 4\u0026deg;C until sprouting. Once sprouts developed, they were transferred to soil in controlled-environment chambers for clonal propagation, following the methods described in (S\u0026aacute;nchez et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). To obtain physiologically uniform and tender tissues particularly suitable for pathogen infection, metabolomic and gene expression analysis, the plants were then transferred to tissue culture. Internodal stem sections were excised, surface-sterilized by gentle washing with a bleach solution followed by sterile water, and placed horizontally in sterile containers containing 0.5 \u0026times; Murashige and Skoog (MS) medium (Vollmer et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Subcultures were performed every four weeks to generate sufficient plantlets, which were subsequently transferred back to soil for pathogen infection assays and molecular analyses. The cultivar Pentland Ivory, carrying the \u003cem\u003eNb\u003c/em\u003e resistance gene in the simplex condition (\u003cem\u003eNb nb nb nb\u003c/em\u003e), was self-pollinated to obtain resistant (\u003cem\u003eNb\u003c/em\u003e) and susceptible (\u003cem\u003enb\u003c/em\u003e) progeny (Marano et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). These progenies were clonally propagated by rooting apical internode cuttings, as described by S\u0026aacute;nchez et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2010\u003c/span\u003e. Pathogen-free potato plants were maintained in a growth chamber at 20 to 25\u0026deg;C with 60% relative humidity, a 16 h photoperiod and a light intensity of 150 to 200 \u0026micro;E m\u003csup\u003e\u0026ndash;2\u003c/sup\u003es\u003csup\u003e\u0026ndash;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePVX cDNA clones and viral inoculation\u003c/h3\u003e\n\u003cp\u003eThe pPVX204 plasmid (Baulcombe et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), containing the PVX UK3 full-length cDNA fused to the green fluorescent protein (\u003cem\u003eGFP\u003c/em\u003e) gene, was modified to create the PVX ROTH1 strain expressing GFP (PVX ROTH1-GFP). This was accomplished by replacing the DNA fragment positioned between the unique \u003cem\u003eBamH\u003c/em\u003eI and \u003cem\u003eApa\u003c/em\u003eI restriction sites in PVX-GFP with the corresponding fragment from the PVX ROTH1 plasmid (Malcuit et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), which includes the 5\u0026rsquo; terminal region of the gene encoding the 25 kDa movement protein (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). The resulting plasmid was designated PVX ROTH1. The functionality of the PVX ROTH1-GFP infectious clone was evaluated by agroinfiltration in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves using mechanical inoculation. For each plant, 10 \u0026micro;g of plasmid DNA (pPVX ROTH1-GFP) was applied in a phosphate buffer (10 mM, pH 7.2) using gentle abrasion. At 10 days post-inoculation (dpi), successful infection was confirmed by the presence of green fluorescence in symptomatic leaves under ultraviolet (UV) illumination, in addition to characteristic PVX-induced symptoms such as mosaic and leaf curling observed in systemic tissues (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). A homogenate inoculum containing infectious virions was prepared from PVX ROTH1-GFP infected \u003cem\u003eN. benthamiana\u003c/em\u003e leaves, following the protocol described by Garcia et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e. Crude sap was adjusted to a concentration of approximately 0.5-1 infective virions/\u0026micro;L and used to inoculate different potato cultivars (Malcuit et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). A randomized experimental design was employed, including eight to ten biological replicates per cultivar. Plants were maintained under controlled environmental conditions, as described previously. Control plants were mock-inoculated with potassium phosphate buffer (10 mM, pH 7.2) only. The progression of local infection was monitored over a 14-day period using a stereomicroscope (BH2; Olympus, Tokyo, Japan).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAgrobacterium-\u003c/b\u003e \u003cb\u003emediated transient expression.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePotato plants at the 4\u0026ndash;5 leaf stage were inoculated by pressure infiltration with \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain C58C1, transformed with pBIN25K1 or pBIN25K3 plasmids (S\u0026aacute;nchez et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). \u003cem\u003eAgrobacterium\u003c/em\u003e cells were cultured in 5 ml of L medium (Sambrook, J.; Fritsch, E.F.; Maniatis \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), supplemented with kanamycin (50 \u0026micro;g/mL) and tetracycline (5 \u0026micro;g/mL), and incubated at 28\u0026deg;C to saturation for 16 h. After centrifugation at 750 \u0026times; g, cells were resuspended in infiltration buffer containing 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 mM morpholineethanesulfonic acid (MES, pH 5.6), and 100 \u0026micro;M acetosyringone (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), and the optical density (OD\u003csub\u003e600\u003c/sub\u003e) was adjusted to 0.8. The cultures were incubated in dark at room temperature for 3 hours with gentle shaking prior to agroinfiltration. Plants were monitored for up to 3 days at 22\u0026deg;C and 50\u0026ndash;60% relative humidity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eZoospore inoculum preparation, infection assays, and lesion quantification of\u003c/b\u003e \u003cb\u003eP. infestans\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eP. infestans\u003c/em\u003e isolate PiNSL-19, classified as genotype EU_2_A1 genotype (Ju\u0026aacute;rez, M.; Azcue, J.; Cano Mogrovejo, L.; Lamour, K.; Bravo-Almonacid, F.F.; Lucca, M.F.; Segretin 2024), was originally obtained from symptomatic cultivar Spunta tubers collected in San Luis Province, Argentina. Cultures were maintained on rye sucrose agar (RSA) medium, prepared according to (Van West et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Briefly, 60 g of rye grains were surface-sterilized in 0.25% sodium hypochlorite for 4 min, rinsed, and incubated at 25\u0026deg;C for 24 h to promote germination. The grains were then dried at 55\u0026deg;C for 3 h, ground, and used to prepare RSA medium by adding 20 g of sucrose and 15 g of agar to a final volume of 1 L. The medium was autoclaved, and cultures were initiated from mycelial discs cryopreserved in 5% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen (PW 1988). Active cultures were maintained at 4\u0026deg;C and subculture weekly. For zoospore production, \u003cem\u003eP. infestans\u003c/em\u003e was grown on RSA plates at 18\u0026deg;C in the dark for 11\u0026ndash;14 days (Van West et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Sporulating cultures were flooded with 3\u0026ndash;5 mL of chilled sterile water and incubated at 4\u0026deg;C for 1\u0026ndash;2 h to stimulate zoospore release. Zoospore concentrations were monitored every 30 min until reaching the concentration of use (2\u0026times;10⁴ \u0026minus;\u0026thinsp;3\u0026times;10⁴ zoospores/mL). The suspension was then transferred to microcentrifuge tubes and maintained at 4\u0026deg;C during inoculation procedures.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e infection assays were conducted using plantlets of potato cultivars Innovator, Kennebec, Spunta and Frital, grown on MS medium supplemented with 2% (w/v) sucrose. A 10 \u0026micro;L droplet of zoospore suspension was applied to the leaf surface, following the protocol of (Huang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Disease progression was documented at 5 dpi via digital imaging. Detached leaf assays were performed on leaves from 4-to 5-week-old soil-grown plants of potato cultivars. Plant growth and infection assays were conducted in parallel at two independent laboratories, INGEBI-CONICET (Buenos Aires) and IBR-CONICET (Rosario). In both facilities, plants were cultivated in growth chambers under standardized conditions (24\u0026deg;C, 16 h light/8 h dark photoperiod, 60% relative humidity). Experimental procedures were independently replicated at both sites to ensure reproducibility. The two youngest fully expanded compound leaves were detached and placed abaxial side up on water-statured floral foam blocks arranged in plastic trays. Each leaflet was then inoculated on the abaxial surface with a 10 \u0026micro;L drop of zoospore suspension. Control leaflets were inoculated with sterile water. After inoculation, the trays were sealed with transparent plastic film to maintain high humidity and prevent desiccation. The inoculated leaves were incubated under the same controlled growth chamber conditions described above.\u003c/p\u003e \u003cp\u003eLesion development was assessed up to 5 dpi, following the protocol by Gabriel et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2007\u003c/span\u003e. Both adaxial and abaxial leaf surfaces were photographed under visible light, and the abaxial surface was additionally photographed under UV light (365 nm) to visualize necrotic areas. Lesion area was quantified using ImageJ software by adjusting brightness, contrast and color saturation parameters with the \u0026ldquo;Adjust-Color Threshold\u0026rdquo; function, following a protocol adapted from the UF IFAS Horticultural Crop Physiology Lab (Agehara et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Leaf and lesion areas were measured for each leaflet. In cases of overlapping lesions, the midrib was used as a boundary to define lesion margins. Proportion of lesion area was calculated as the ratio of lesion area and total leaf area.\u003c/p\u003e\n\u003ch3\u003eRosemary extract preparation, plant treatment and pathogen inoculation\u003c/h3\u003e\n\u003cp\u003eLyophilized aqueous rosemary extract (ARE) was prepared as described by Martin et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e. The extract was standardized to a final rosmarinic acid (RA) concentration of approximately 400 \u0026micro;M, and all treatments were quantified based on RA content. Although RA is the major phenolic component, ARE displays stronger protective activity than RA alone, suggesting the contribution of additional bioactive compounds (Martin et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Four-week-old soil-grown potato plants were treated by spraying 100 \u0026micro;L of ARE onto the adaxial surface of one fully expanded leaf per plant, once daily for two consecutive days. Control plants were similarly treated with water. Pathogen inoculation was performed 24 h after the second application (Fig. S2). Lesion area associated to \u003cem\u003eP. infestans\u003c/em\u003e infection was quantified using Image J as indicated above. Data were modeled using a beta distribution within a randomized block design framework, suitable for proportion data derived from normally distributed variables. The experiment day was treated as a random variable. Statistical analyses were performed in RStudio (R version 4.0.1) using ggplot2 and RColorBrewer for visualization, glmmTMB for Beta model fitting, DHARMa for model assumption checks, and emmeans for post-hoc comparisons.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSample preparation for proton nuclear magnetic resonance (H NMR) spectroscopy\u003c/h3\u003e\n\u003cp\u003eLeaf samples were prepared for metabolite analysis following the protocol described by Kim et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e. A total of 20 leaf extract samples (five per cultivar) were analyzed. Apical leaves (~\u0026thinsp;200 mg) from plants at the 3\u0026ndash;4 leaf stage were collected, excluding petioles, snap-frozen in liquid nitrogen, and homogenized using a tissue disruptor (MM-400, Restch GmbH, Haan, Germany). Homogenates were lyophilized, and ~\u0026thinsp;30 mg of the resulting dry powder was subjected to polar metabolite extraction using 750 \u0026micro;L of phosphate buffer (90 mM, pH 6.0) prepared in deuterium oxide (D\u003csub\u003e2\u003c/sub\u003eO) and containing 0.1% (w/v) 3-(trimethylsilyl)-2,2,3,3-d4-propionic acid (TSP) as an internal standard. Samples were vortexed for 2 min at room temperature and sonicated three times (5 min each, with alternating 30 s on/off pulses) in an ultrasonic bath (Branson SS10 EMT, Brookfield, Connecticut, USA). Following centrifugation at 25,000 \u0026times; g for 30 min at 4\u0026deg;C, 500 \u0026micro;L of clarified supernatant was transferred to 5 mm NMR tubes (Wilmad NMR, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) for spectral analysis. This methodology allows to simultaneously detect primary (sugars, organic acids, amino acids) and secondary metabolites (flavonoids, alkaloids, terpenoids), providing quantitative and structural information.\u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH NMR spectra were obtained at 300 K on a Bruker Avance III 700-MHz NMR spectrometer (Bruker Biospin, Rheinstetten, Germany) equipped with a 5-mm TXI probe. One-dimensional \u003csup\u003e1\u003c/sup\u003eH NMR spectra of leaf extract were acquired using a standard 1-D NOESY pulse sequence (noesygppr1d) with water presaturation. The mixing time was set to 10 ms, the acquisition time to 2.228 s, and the relaxation delay to 4 s. Spectra were acquired using four dummy scans and 32 acquisition scans, with 64 K time domain points and a spectral width of 20 ppm. Free induction decays were multiplied by an exponential window function with a line broadening factor of 0.3 Hz prior to Fourier transformation.\u003c/p\u003e\n\u003ch3\u003eData pre-processing and multivariate analysis of H NMR spectra\u003c/h3\u003e\n\u003cp\u003eSpectroscopic data were processed in MATLAB (version R2015b, The MathWorks). Spectra were referenced to TSP at 0.0 ppm, followed by baseline correction and phase adjustment using custom functions developed at Imperial College London (provided by T. Ebbels and H. Keun). Each spectrum was segmented into integrated regions (\u0026ldquo;bins\u0026rdquo;) of equal width (0.04 ppm, standard bucket width). Non-informative regions containing no metabolite signals, including the TSP signal and the water signal resonance region (between 4.9 and 4.6 ppm), were excluded. Spectra were normalized using the probabilistic quotient (PQN) method (Dieterle et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Alignment was performed using the recursive segment-wise peak alignment (RSPA) algorithm within user-defined spectral windows (Veselkov et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Spectral data matrices were subjected to multivariate curve resolution-alternating least squares (MCR-ALS) in MATLAB\u0026reg;, with spectra segmented into windows containing relevant signals, and non-negativity constraints were applied. The optimal number of components was determined, and integrated signals were exported for further matrix construction. The resulting data matrix was imported into MetaboAnalyst 6.0 software (Pang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) for multivariate statistical analysis (Table S2). Data were centered and scaled using unit variance (UV). Principal component analysis (PCA) was performed to explore sample distribution, and orthogonal partial least squares discriminant analysis (OPLS-DA) was applied to maximize group separation and identify discriminant signals. Supervised models were validated using 200 permutation tests. Discriminant variables, showing significant differences, were identified through a Volcano plot, applying a fold change threshold of 2 and a significance level of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Only the discriminant signals were subsequently assigned, ensuring that the interpretation focused exclusively on the features that contributed meaningfully to group separation. Discriminant signals were annotated using the Biological Magnetic Resonance Data Bank (BMRB) and the Human Metabolome Database (HMDB) (Wishart et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction, reverse transcription, and quantitative PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from three leaf disks (~\u0026thinsp;100 mg of fresh tissue) per cultivar using the NucleoSpin\u0026reg; RNA kit (Macherey-Nagel, D\u0026uuml;ren, Germany), according to the manufacturer instructions. Reverse transcription was performed with RevertAid (ThermoFisher Scientific, Waltham, Massachusetts, USA). For virus quantification, 1 ng of DNase-treated RNA was used, whereas 1 \u0026micro;g of total RNA was employed for the analysis of plant gene expression. In both cases, oligo (dT)\u003csub\u003e12\u0026ndash;18\u003c/sub\u003e primers were used to synthesize first-strand complementary DNA (cDNA). Quantitative PCR (qPCR) reactions were carried out in a final volume of 20 \u0026micro;L, containing 1 \u0026micro;L of EvaGreen dye (Biotium, Fremont, California, USA), 5\u0026ndash;10 pmol of each primer, 3 mM MgCl₂, cDNA (1:10 dilution), and 1 U Taq DNA polymerase (PBL, Buenos Aires, Argentina). Amplification was monitored using the Mastercycler\u0026reg; ep realplex system (Eppendorf, Hamburg, Germany). Primer sequences targeting the PVX ROTH1 coat protein (\u003cem\u003eCP\u003c/em\u003e) and plant defense-related genes, including \u003cem\u003ePhenylalanine Ammonia-Lyase\u003c/em\u003e (\u003cem\u003ePAL\u003c/em\u003e), \u003cem\u003eIsochorismate Synthase 1\u003c/em\u003e (\u003cem\u003eICS1\u003c/em\u003e), \u003cem\u003ePathogenesis-Related Protein 1\u003c/em\u003e (\u003cem\u003ePR1\u003c/em\u003e), \u003cem\u003eUDP-glucosyltransferase 74B1 (UGT74B1\u003c/em\u003e), \u003cem\u003eAllene Oxidase Synthase 2\u003c/em\u003e (\u003cem\u003eAOS2\u003c/em\u003e), \u003cem\u003eAcyl-coenzyme A oxidase (ACX1)\u003c/em\u003e, \u003cem\u003eAcyl-coenzyme A oxidase (ACX3)\u003c/em\u003e, and \u003cem\u003eElongation Factor 1-α\u003c/em\u003e (\u003cem\u003eEF-α\u003c/em\u003e), as well as thermal cycling conditions are provided in Table S3. Relative transcript levels were calculated using the ΔΔCt method (Livak and Schmittgen \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Gene expression data were normalized against \u003cem\u003eEF-α\u003c/em\u003e transcript levels. For viral \u003cem\u003eCP\u003c/em\u003e RNA quantification, samples taken at 24 hours post inoculation (hpi) were used as reference. For plant gene expression analysis in non-inoculated plants, samples from cultivar Spunta served as reference. In the priming experiments, PVX ROTH1 \u003cem\u003eCP\u003c/em\u003e RNA and plant defense marker genes were analyzed at 24 and 48 hpi. Water treated plants at each time served as reference. Each experiment included four biological replicates per cultivar and, each comprising pool material from two to four leaves collected from independent plants. Assays were independently repeated three times. Statistical significance was performed using one-way ANOVA (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eBasal metabolic profiles of potato cultivars to evaluate intrinsic defense capacity\u003c/h2\u003e \u003cp\u003eConstitutive metabolic traits can strongly influence how plants respond to environmental challenges and pathogen attacks, ultimately shaping their resilience under changing climatic conditions (Salam et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). To investigate whether such traits underlie differences in susceptibility to PVX and \u003cem\u003eP. infestans\u003c/em\u003e, leaves from the cultivars Innovator, Kennebec, Spunta, and Frital were analyzed using \u0026sup1;H NMR spectroscopy. This approach established metabolic baselines for each cultivar, providing a framework to assess constitutive defense capacity and the potential impact of defense-priming strategies in susceptible genotypes.\u003c/p\u003e \u003cp\u003eAcross the four cultivars, 270 metabolic features were detected, with 197 assigned to known metabolites. Among these, 11 metabolites were annotated and 9 identified as discriminant variables, including the alkaloid trigonelline, the amino acid asparagine, the indole derivative 3-indolylmethyl, and the fatty acid linoleic acid (Table S2). Principal component analysis (PCA) of the 197 integrated \u0026sup1;H NMR peaks explained 63.2% of the total variance, with PC1-PC4 contributing 27.2%, 15.1%, 11.8%, and 9.1%, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, PC1 and PC3 clearly separated Innovator from the other cultivars, reflecting its distinct constitutive metabolic profile associated with resistance. PC2 and PC4 distinguished Kennebec and Spunta, whereas Frital overlapped with both susceptible cultivars.\u003c/p\u003e \u003cp\u003eThe obtained metabolic profiles align with previously reported resistance loci (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e): Innovator carries multiple \u003cem\u003eRpi-R\u003c/em\u003e genes and the \u003cem\u003eNb\u003c/em\u003e gene, consistent with resistance to \u003cem\u003eP. infestans\u003c/em\u003e and PVX strain ROTH1 (PVX ROTH1), respectively. In contrast, both Kennebec and Spunta lack \u003cem\u003eNb\u003c/em\u003e and contain only \u003cem\u003eRpi-R1\u003c/em\u003e, which confers limited or no protection against virulent \u003cem\u003eP. infestans\u003c/em\u003e isolates carrying non-recognized Avr1 variants (Coomber et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Consequently, both cultivars are likely susceptible to PVX ROTH1 and \u003cem\u003eP. infestans\u003c/em\u003e. Frital presents a less defined genetic background, suggesting a potential intermediate or susceptible phenotype. Based on these genetic backgrounds, we categorized the cultivars into putative resistant- or susceptible-like groups.\u003c/p\u003e \u003cp\u003eTo investigate metabolic differences between potentially resistant- and susceptible-like cultivars, supervised OPLS-DA was performed comparing Innovator with Kennebec and Spunta, while Frital was excluded from subsequent binary analysis. This approach revealed clear separation between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). A heat map of the 30 most abundant metabolites across five biological replicates per cultivar highlighted specific metabolic signatures and facilitated direct comparison of metabolite abundance between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Differential metabolite accumulation between resistant- and susceptible- groups was further assessed using volcano plot analysis (fold change\u0026thinsp;\u0026gt;\u0026thinsp;2.0; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), which identified metabolites with statistically significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). In the resistant-like group, 3-indolylmethyl, trigonelline, linoleic acid (or a related derivative), and two unidentified signals (U_32_1 and U_87_2) were upregulated, whereas asparagine and three unidentified signals (U_33_4, U_38_2, and U_115_1) were downregulated. Interestingly, among the susceptible-like cultivars, Spunta showed the lowest metabolic abundance (Fig. S3a).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together, untargeted NMR analyses revealed cultivar-specific metabolic signatures. Innovator was enriched in trigonelline, linoleic acid and indole-derived metabolites, reflecting a constitutive metabolic state that may support basal pathogen defense. In contrast, Kennebec and Spunta displayed a narrower and less diverse metabolic profile, particularly in compounds potentially associated with basal defense, suggesting a metabolic context less favorable for early stress responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eConstitutive expression of defense-related genes in potato cultivars\u003c/h2\u003e \u003cp\u003eBasal defense responses were also assessed by quantifying the expression of four candidate defense genes in non-inoculated leaves of Innovator and Spunta, selected as representative cultivars with contrasting resistance loci and constitutive metabolic profiles identified in the metabolomic analysis. These genes were \u003cem\u003ePAL1\u003c/em\u003e, encoding phenylalanine ammonia-lyase, a pivotal enzyme in phenylpropanoid biosynthesis; \u003cem\u003eICS\u003c/em\u003e, encoding isochorismate synthase, essential for salicylic acid (SA) production; \u003cem\u003ePR1\u003c/em\u003e, a canonical SA-responsive pathogenesis-related gene; and \u003cem\u003eUGT74B1\u003c/em\u003e, a putative UDP-glycosyltransferase involved in indolic glucosinolate glycosylation (Vleeshouwers et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Navarre and Mayo \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Grubb et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Notably, \u003cem\u003ePR1\u003c/em\u003e expression was markedly elevated in Innovator (19.52\u0026thinsp;\u0026plusmn;\u0026thinsp;10.71 vs. 1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13), a widely used marker of SA-dependent defense signaling, while \u003cem\u003eUGT74B1\u003c/em\u003e showed a moderate but consistent increase (3.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63 vs. 1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34). In contrast, \u003cem\u003ePAL1\u003c/em\u003e and \u003cem\u003eICS\u003c/em\u003e expression levels were comparable between cultivars (Fig. S3b). These patterns, together with elevated tryptophan-derived metabolites in Innovator, indicate a constitutive defense state that may contribute to its enhanced pathogen resistance.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCultivar-specific potato defenses against PVX ROTH1 and\u003c/b\u003e \u003cb\u003eP. infestans\u003c/b\u003e \u003cb\u003eidentify candidates for ARE priming\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIntegrated infection assays were performed to validate the predicted resistance/susceptibility profiles and to investigate how constitutive metabolic and transcriptional signatures shape cultivar-specific defense. Experiments were accomplished using PVX ROTH1 and a locally isolated \u003cem\u003eP. infestans\u003c/em\u003e strain. This experimental framework provides evidence to identify and confirm susceptible cultivars suitable for defense reinforcement via ARE-mediated priming.\u003c/p\u003e \u003cp\u003eTo characterize antiviral responses, leaves from each cultivar (Innovator, Frital, Kennebec, and Spunta) were mechanically inoculated with a GFP-expressing PVX ROTH1 clone (PVX ROTH1-GFP). Pentland Ivory, which carries the \u003cem\u003eNb\u003c/em\u003e resistance gene, and a susceptible \u003cem\u003enb\u003c/em\u003e genotype derived from selfing were included as controls for PVX resistance and susceptibility, respectively (Malcuit et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Marano et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Fluorescence imaging revealed restricted viral replication in Innovator and Frital, where GFP signals were confined to the inoculation site and associated with localized hypersensitive response (HR) lesions. This pattern is consistent with \u003cem\u003eNb\u003c/em\u003e-mediated recognition of the viral PVX 25-kDa movement protein (25K1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In contrast, Kennebec and Spunta exhibited extensive GFP fluorescence spreading beyond the inoculation site, reflecting a susceptible phenotype with unrestrained viral progression. Quantification of PVX CP RNA at 48 hpi corroborated these observations: Innovator and Frital showed CP transcript levels of 1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44- and 1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51-fold relative to the resistant \u003cem\u003eNb\u003c/em\u003e control, respectively. Conversely, Kennebec and Spunta exhibited significant increases of 10.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.63- and 18.17\u0026thinsp;\u0026plusmn;\u0026thinsp;7.36-fold, respectively, while the susceptible \u003cem\u003enb\u003c/em\u003e control displayed the highest accumulation (36.3\u0026thinsp;\u0026plusmn;\u0026thinsp;13.7-fold relative to \u003cem\u003eNb\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe \u003cem\u003eNb\u003c/em\u003e-mediated resistance was subsequently confirmed by agroinfiltration assays expressing either the 25K1 effector or the homologous 25K3 from PVX UK3. At 72 hpi, Innovator and Frital developed localized HR upon 25K1 expression\u0026mdash;mirroring the \u003cem\u003eNb\u003c/em\u003e-positive control\u0026mdash;whereas no HR was observed in Kennebec, Spunta, or any cultivar expressing 25K3 (Fig. S4a). Genotyping with SPUD237 and SPUD839 markers further verified the presence of the \u003cem\u003eNb\u003c/em\u003e gene in Innovator and Frital and its absence in Kennebec and Spunta (Fig. S4b). Collectively, these results demonstrate for the first time that Frital possesses resistance to PVX ROTH1 similar to Innovator, while Kennebec and Spunta exhibit a susceptible phenotype.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate cultivar-specific defense against oomycetes, \u003cem\u003ein vitro\u003c/em\u003e propagated plants were inoculated with \u003cem\u003eP. infestans\u003c/em\u003e isolate PiNSL-19. Innovator developed localized HR lesions that restricted pathogen spread and sporulation, whereas Spunta and Kennebec showed severe disease symptoms. Frital exhibited an intermediate phenotype, with lesion development falling between the resistant and susceptible cultivars. These responses were recapitulated in detached leaves from soil-grown plants: Innovator restricted lesion expansion, while Spunta and Kennebec developed large, sporulating lesions. Lesion areas were quantitatively assessed at 4 dpi and normalized to Spunta (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,b).\u003c/p\u003e \u003cp\u003eTogether, these results confirm that cultivar-specific responses to PVX and \u003cem\u003eP. infestans\u003c/em\u003e are consistent across infection assays, reflecting the combined action of basal and effector-triggered immunity. The strong correlation between molecular markers and observed phenotypes supports the use of genetic screening to streamline the identification of susceptible cultivars. Consequently, this approach integrates constitutive and inducible defense mechanisms to target cultivars for ARE-mediated resistance reinforcement under climate-driven pathogen pressure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eARE treatment primes potato defense responses against viral and oomycete pathogens\u003c/h2\u003e \u003cp\u003ePrevious studies demonstrated that ARE functions as an effective priming agent in diverse plant species, including \u003cem\u003eN. tabacum\u003c/em\u003e, \u003cem\u003eGlycine max\u003c/em\u003e, and \u003cem\u003eCitrus limon\u003c/em\u003e. This treatment enhances resistance against a wide range of pathogens, including viruses, bacteria and fungi, by reinforcing basal immune signaling and promoting a faster activation of inducible defense pathways upon infection (Martin et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). To evaluate the potential of ARE to prime inducible defenses against both PVX and \u003cem\u003eP. infestans\u003c/em\u003e, the highly susceptible cultivar Spunta, was selected. This genotype, which lacks effective genetic resistance mechanisms to multiple pathogens, provides an appropriate model to assess the efficacy of ARE-mediated priming. In parallel, the PVX ROTH1-resistant cultivar P. Ivory (carrying the \u003cem\u003eNb\u003c/em\u003e gene) and the susceptible \u003cem\u003enb\u003c/em\u003e genotype were included as controls, allowing comparison between ARE-induced responses and those conferred by genetic resistance.\u003c/p\u003e \u003cp\u003ePlants were treated twice with 100 \u0026micro;L of ARE at 24 h intervals, and 24 h after the last application, leaves were inoculated with PVX ROTH1-GFP, and local infection symptoms were monitored for a 10-day period. Viral movement and replication were monitored by fluorescence microscopy and qPCR analysis of \u003cem\u003eCP\u003c/em\u003e-PVX levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b). In Spunta, ARE treatment restricted viral cell-to-cell movement and significantly reduced viral accumulation compared to water-treated controls at 24 and 48 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b). A similar decrease in \u003cem\u003eCP\u003c/em\u003e-PVX transcription levels was observed in the PVX ROTH1-susceptible cultivar P. Ivory, indicating that both Spunta and this genotype responded comparably to ARE treatment in the absence of functional \u003cem\u003eNb\u003c/em\u003e resistance gen. qPCR analysis showed an 80\u0026ndash;90% reduction in \u003cem\u003eCP\u003c/em\u003e-PVX transcript levels at 24 and 48 hpi in ARE-treated susceptible cultivars. In contrast, in the resistant \u003cem\u003eNb\u003c/em\u003e genotype, which inherently restricts PVX ROTH1 accumulation through HR, ARE treatment further reduced fluorescence intensity at 48 hpi, suggesting an additive priming effect. Fluorescence was only observed in the area surrounding the initial infection site to accurately assess viral spread within the tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Accordingly, \u003cem\u003eCP\u003c/em\u003e-PVX transcript levels remained consistently low regardless of treatment, likely due to the strong immune response mediated by \u003cem\u003eNb\u003c/em\u003e-dependent resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Together, these results demonstrate that ARE limits PVX ROTH1 replication in susceptible cultivars and can further potentiate antiviral defenses even in the presence of functional resistance genes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on prior studies in \u003cem\u003eN. tabacum\u003c/em\u003e infected with tobacco necrosis virus strain A (TNVA), where ARE treatment enhanced SA accumulation and defense gene expression (Martin et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), we hypothesized that ARE may similarly prime SA-mediated defenses in potato against PVX ROTH1. To test this, we analyzed the expression of key SA-related genes involved in biosynthesis (\u003cem\u003ePAL, ICS1\u003c/em\u003e) and downstream signaling (\u003cem\u003ePR1\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eIn Spunta, ARE treatment induced a delayed upregulation of \u003cem\u003eICS1\u003c/em\u003e transcripts at 48 hpi, without significantly modulation of \u003cem\u003ePAL\u003c/em\u003e or \u003cem\u003ePR1\u003c/em\u003e expression, suggesting a temporally limited activation of the isochorismate- dependent SA biosynthetic pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). For comparison, the control cultivar Pentland Ivory, carrying either the resistant (\u003cem\u003eNb\u003c/em\u003e) or susceptible (\u003cem\u003enb\u003c/em\u003e) alleles, was also analyzed. In these control genotypes, ARE treatment triggered earlier and substantially stronger transcriptional activation of SA-associated genes. Specifically, \u003cem\u003ePAL\u003c/em\u003e and \u003cem\u003ePR1\u003c/em\u003e, were significantly upregulated at 24 and 48 hpi in both genotypes. Interestingly, ARE-treated \u003cem\u003enb\u003c/em\u003e plants reached \u003cem\u003ePAL\u003c/em\u003e and \u003cem\u003ePR1\u003c/em\u003e expression levels comparable to those of the resistant \u003cem\u003eNb\u003c/em\u003e genotype, suggesting that ARE can enhance SA-dependent defense activation even in the absence of a functional \u003cem\u003eNb\u003c/em\u003e resistance gene. This differential response, stronger in Pentland Ivory than in Spunta, highlights a genotype-dependent predisposition to ARE-mediated priming, whereby certain genetic backgrounds may exhibit an enhanced capacity to activate defense signaling independently of effector-specific recognition.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003e. \u003cb\u003eAqueous rosemary extract (ARE) confers protection against PVX ROTH1-GFP in potato cultivars. a.\u003c/b\u003e Potato leaves of cultivars Pentland Ivory (\u003cem\u003eNb\u003c/em\u003e and \u003cem\u003enb\u003c/em\u003e) and Spunta (Sp) were treated with aqueous rosemary extract (ARE) or water, and then inoculated with PVX ROTH1-GFP. Leaves were imaged by laser scanning confocal microscopy at 2 days post-inoculation (dpi) in resistant and 4 dpi in susceptible cultivars. \u003cb\u003eb.\u003c/b\u003e Reverse transcription and quantitative PCR (qPCR) analysis at 24 and 48 h post-inoculation (hpi) in PVX ROTH1-GFP-inoculated plants treated with ARE or water. Values were normalized to the constitutive \u003cem\u003eEF-α\u003c/em\u003e gene and fold changes were expressed relative to inoculated water-treated leaves (controls) of Pentland Ivory \u003cem\u003enb\u003c/em\u003e genotype (\u003cem\u003enb\u003c/em\u003e) for each time. Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;4 replicates, each consisting of four leaves from four different plants) are shown. Different letters indicate significant differences at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (one-way ANOVA, Tukey\u0026rsquo;s test). \u003cb\u003ec.\u003c/b\u003e qPCR analysis of \u003cem\u003ePAL\u003c/em\u003e, \u003cem\u003eIC\u003c/em\u003e1, and \u003cem\u003ePR1\u003c/em\u003e at 24 and 48 hpi with PVX ROTH1-GFP in potato cultivars treated with ARE or water. Values were normalized to the constitutive \u003cem\u003eEF-α\u003c/em\u003e gene and fold changes were expressed relative to inoculated water-treated leaves (control) at each time. Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of 5 replicates, each consisting of five leaves from five different plants are shown. Asterisks indicate significant differences at *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008 (one-way ANOVA, Tukey\u0026rsquo;s test). \u003cem\u003eEF-α\u003c/em\u003e, \u003cem\u003eELONGATION FACTOR 1-α\u003c/em\u003e; \u003cem\u003ePAL\u003c/em\u003e, \u003cem\u003ePHENYLALANINE AMMONIA-LYASE\u003c/em\u003e; \u003cem\u003eICS\u003c/em\u003e, \u003cem\u003eISOCHORISMATE SYNTHASE\u003c/em\u003e; \u003cem\u003ePR1\u003c/em\u003e, \u003cem\u003ePATHOGENESIS-RELATED PROTEIN 1\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eFor \u003cem\u003eP. infestans\u003c/em\u003e infection assays, potato plants of the highly susceptible cultivar Spunta were treated twice with 100 \u0026micro;L of ARE. Twenty-four hours after the second application, detached leaves were inoculated with the virulent isolate PiNSL19 (Fig. S2). Disease progression was monitored over 5-days based on lesion development and expansion. At 72 hpi, ARE-treated leaves showed smaller and lighter necrotic lesions compared with the larger, darker ones in water-treated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and Fig. S5a). Quantitative image analysis indicated a 30% reduction in lesion size and overall disease severity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and Fig. S5b). \u003cem\u003eIn vitro\u003c/em\u003e diffusion assays confirmed that ARE had no direct effect on \u003cem\u003eP. infestans\u003c/em\u003e mycelial growth, suggesting that protection was associated with activation of host defenses rather than antifungal activity (\u003cem\u003edata not shown\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDefense against \u003cem\u003eP. infestans\u003c/em\u003e relies on coordinated hormonal regulation, with SA responses dominating the early biotrophic phase and jasmonic acid (JA) contributing during the necrotrophic stage (Halim et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). To explore whether ARE affected these pathways, we analyzed the expression of representative marker genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). All three SA-associated genes were induced at 72 hpi, but no significant differences were detected between ARE- and water-treated plants, indicating that ARE does not substantially enhance SA-mediated defenses under the tested conditions. For the JA pathway, \u003cem\u003eAOS2\u003c/em\u003e involved in early JA biosynthesis, and \u003cem\u003eACX1\u003c/em\u003e and \u003cem\u003eACX3\u003c/em\u003e, encoding acyl-CoA oxidases of the β-oxidation pathway, were selected based on previous transcriptomic data from Spunta infected with PiNSL19 (Juarez \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Juarez et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). \u003cem\u003eAOS2\u003c/em\u003e expression increased at 48 hpi in both treatments, whereas \u003cem\u003eACX1\u003c/em\u003e and \u003cem\u003eACX3\u003c/em\u003e transcripts were consistently lower in ARE-treated plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). These findings indicate that ARE enhances Spunta resistance to \u003cem\u003eP. infestans\u003c/em\u003e, accompanied by moderate activation of SA- and JA-related genes. The magnitude and timing of these responses suggest that additional, genotype-dependent regulatory pathways may also contribute to the observed protection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eUnderstanding how potato cultivars respond to pathogens under fluctuating environmental conditions is critical, as genetic resistance is often incomplete or can be overcome by emerging threats. ARE, a plant-derived extract with broad-spectrum priming activity, can enhance plant defense responses against such challenges. In this work, we evaluated ARE as a potential priming agent in potato by analyzing pathogen-free tubers to identify cultivars with contrasting susceptibility based on constitutive metabolomic profiles and defense marker gene expression.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMolecular signatures of constitutive resistance in Innovator\u003c/h2\u003e \u003cp\u003eThis analysis revealed distinct cultivar-specific metabolic states, with Innovator consistently separating from susceptible backgrounds. Innovator leaves accumulated higher levels of trigonelline, linoleic acid-related signals, tryptophan-derived indolic metabolites, and several unassigned features, whereas susceptible cultivars such as Spunta showed elevated asparagine and other low molecular weight signals. Notably, the five unassigned signals (U_32_1, U_87_2, U_33_4, U_38_2, and U_115_1) may represent yet undescribed metabolites with potential as novel chemical indicators of basal resistance.\u003c/p\u003e \u003cp\u003eThe differential accumulation of these compounds, in parallel with increased transcript abundance of SA-responsive markers such as \u003cem\u003ePR1\u003c/em\u003e, support a constitutively primed metabolic state likely contributing to high resistance in Innovator. Among these features, trigonelline (N-methylnicotinate) emerges as a particularly interesting candidate. In potato, ¹H NMR-based metabolomics revealed trigonelline accumulation in SAMDC (S-adenosylmethionine decarboxylase) transgenic lines, with moderate levels linked to protective functions (Defernez et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Its primary synthesis in green tissues, potentially linked to chloroplast metabolism, together with its capacity to be recycled into nicotinic acid for NAD biosynthesis, suggests additional roles in metabolite recycling, detoxification, and cellular regulation (Katahira and Ashihara \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Beyond its central metabolic role, trigonelline has emerged as a metabolite consistently linked to biotic defense. In tomato, its accumulation is triggered by \u003cem\u003eAlternaria alternata\u003c/em\u003e infection or chitin treatment, where it contributes to pattern-triggered immunity by inhibiting fungal growth at physiologically relevant concentrations (Muñoz Hoyos et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Similarly, recent NMR-based metabolomic analysis in tomato brown rugose fruit virus (ToBRFV)-infected plants showed strong trigonelline accumulation, further reinforcing its role as a defense-associated metabolite across plant species (Salmerón et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Collectively, these findings position trigonelline at the interface of primary metabolism and inducible defense, with its constitutive accumulation in Innovator likely contributing to a primed resistance state. In parallel, basal enrichment of linoleic acid, a polyunsaturated fatty acid, underscores lipid remodeling as a complementary layer of this primed configuration and links jasmonate biosynthesis with broad stress adaptation (He and Ding \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This defense profile likely reinforces Innovator readiness to respond to environmental challenges. Complementing these results, tryptophan-derived 3-indolylmethyl metabolites, which are linked to glucosinolate pathways in Brassicaceae (Malcuit et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), were also elevated in Innovator, suggesting they may perform similar defense-related functions in Solanaceae. Their accumulation may involve uncharacterized pathways, potentially including glycosyltransferases such as UGT74B1, which was upregulated in Innovator, and may contribute to basal resistance. Consistently, the constitutive elevated levels of \u003cem\u003ePR1\u003c/em\u003e in Innovator support the involvement of SA-associated defense signaling in basal immunity, in line with previous reports linking basal \u003cem\u003ePR\u003c/em\u003e expression to partial resistance, particularly through restricting \u003cem\u003eP. infestans\u003c/em\u003e during the early biotrophic phase of infection (Vleeshouwers et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Navarre and Mayo \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Halim et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Supporting this, early untargeted liquid chromatography–mass spectrometry (LC-MS) metabolomics in the resistant cultivar Ziyun No.1 revealed elevated basal SA levels, phenylpropanoid intermediates, and triterpenoids, indicating metabolite-mediated defense priming (Zhu et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In Innovator, a comparable primed metabolic state may accelerate defense responses and complement effector-triggered immunity for stronger pathogen restriction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eResistance architecture and infection outcomes\u003c/h2\u003e \u003cp\u003eThe differences in immune status defense profile align with the resistance architecture described for these cultivars, as Innovator carries the \u003cem\u003eNb\u003c/em\u003e gene (PVX ROTH1) and \u003cem\u003eRpi-R1\u003c/em\u003e, \u003cem\u003eRpi-R2\u003c/em\u003e-like, \u003cem\u003eRpi-R3a\u003c/em\u003e and \u003cem\u003eRpi-R3b\u003c/em\u003e (\u003cem\u003eP. infestans\u003c/em\u003e), whereas Spunta lacks functional resistance determinants. Results from the PVX ROTH1 and PiNSL-19 infection assays corroborated the observed defense profiles. Interestingly, Notably, while the PiNSL-19 genome is devoid of \u003cem\u003eAvr1\u003c/em\u003e, it encompasses several homologs of the \u003cem\u003eAvr2\u003c/em\u003e gene family (Jaurez 2024, Juarez et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and the isolate was unable to overcome Innovator, indicating that at least one corresponding R–Avr interaction remains functional. By contrast, although Spunta cultivar carries \u003cem\u003eRpi-R1\u003c/em\u003e (Armstrong et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), this resistance is ineffective against virulent isolates in which \u003cem\u003eAvr1\u003c/em\u003e alleles evade recognition, illustrating the limited durability of single-gene-resistance (Coomber et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eARE-mediated defense priming in susceptible potato cultivars\u003c/h2\u003e \u003cp\u003eARE treatment significantly reduces PVX ROTH1 accumulation in Spunta and in the susceptible cultivar Pentland Ivory (genotype \u003cem\u003enb\u003c/em\u003e), correlating with increased transcription of SA-related defense genes (\u003cem\u003ePAL\u003c/em\u003e, \u003cem\u003eICS1\u003c/em\u003e, \u003cem\u003ePR1\u003c/em\u003e). These results align with previous findings in \u003cem\u003eN. tabacum\u003c/em\u003e, where ARE mitigated viral symptoms via redox and hormonal modulation (Martin et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and in \u003cem\u003eS. lycopersicum\u003c/em\u003e, where RA treatment modulated redox signaling and delayed fruit ripening (Zhu et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Notably, ARE also enhanced resistance in the PVX-ROTH1-resistant genotypes carrying \u003cem\u003eNb\u003c/em\u003e, suggesting additive effects with the effector-triggered immunity and highlighting its potential as a priming agent across different genetic backgrounds. The stronger induction of \u003cem\u003ePR1\u003c/em\u003e and \u003cem\u003ePAL\u003c/em\u003e observed in Pentland Ivory is consistent with the central role of SA signaling in antiviral defense (Sánchez et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In potato, endogenous SA levels tightly correlate with basal \u003cem\u003ePR1\u003c/em\u003e expression, a canonical marker of SA-dependent immune activation (Vleeshouwers et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Navarre and Mayo \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Thus, the contrasting transcriptional responsiveness of Spunta and Pentland Ivory likely reflects intrinsic differences in SA pathway capacity, providing a mechanistic basis for the genotype-specific amplitude of ARE-mediated priming during PVX infection. Comparable genotype-dependent variability has been reported for β-aminobutyric acid (BABA), a well-established immune-priming compound. In tomato, BABA induces strong PR1 accumulation and enhances resistance to \u003cem\u003eP. infestans\u003c/em\u003e (Cohen and Gisi \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), nevertheless the magnitude and stability of this response vary considerably depending on plant genotype and on the controlled environmental conditions under which priming is induced (Cohen, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Overall, the data indicate that genotype-dependent differences in SA responsiveness shape both the detectability of PR1 induction and the amplitude of ARE-mediated priming.\u003c/p\u003e \u003cp\u003eIn Spunta, ARE conferred moderate but reproducible protection against \u003cem\u003eP. infestans\u003c/em\u003e, with lesion size reduced by approximately 30%. Notably, this physiological protection was not accompanied by significant changes in the expression of canonical SA- or JA-associated defense genes, consistent with the intrinsically limited SA responsiveness of this cultivar. In our experiments, a clear difference in PR1 induction between water- and ARE-treated plants was observed only at 48 hpi with PVX ROTH1, indicating a delayed transcriptional response consistent with the genotype-specific SA responsiveness of Spunta. Accordingly, ARE conferred measurable protection against both \u003cem\u003eP. infestans\u003c/em\u003e and PVX even in the absence of robust early PR1 activation, suggesting a primed state operating through defense mechanisms not captured by classical hormonal markers, such as altered pathogen perception, reinforcement of physical barriers, or redox-based adjustments. Similar temporal constraints have been described for jasmonate-mediated priming, where early signaling events rapidly occur but transcriptional activation of defense genes is only detectable within a defined time window after stimulus (Arevalo-Marín et al. 2021), highlighting the importance of sampling timing. In summary, ARE primes potato defenses in a genotype-dependent manner, providing measurable protection against PVX and \u003cem\u003eP. infestans\u003c/em\u003e even in the absence of early PR1 induction, and highlighting its potential for enhancing innate immunity also in this crop.\u003c/p\u003e \u003cp\u003eIn summary, ARE primes potato defenses in a genotype-dependent manner, providing measurable protection against PVX and \u003cem\u003eP. infestans\u003c/em\u003e, and highlighting its potential for enhancing innate immunity in this crop.\u003c/p\u003e \u003c/div\u003e "},{"header":"Conclusions and future perspectives","content":"\u003cp\u003eCollectively, these findings demonstrate that priming with natural products, such as ARE, provides a functional bridge between constitutive and inducible resistance mechanisms, thereby complementing innate defense layers. The broad-spectrum effectiveness of ARE across diverse plant species, including \u003cem\u003eNicotiana\u003c/em\u003e, \u003cem\u003eGlycine\u003c/em\u003e, \u003cem\u003eCitrus\u003c/em\u003e, and \u003cem\u003eSolanum\u003c/em\u003e, highlights its considerable versatility as a defense elicitor. Furthermore, the established metabolic and gene expression signatures of the resistant cultivar Innovator offer a robust reference framework for evaluating and optimizing future priming strategies. The observed genotype-dependent differences in SA responsiveness and \u003cem\u003ePR1\u003c/em\u003e inducibility suggest that ARE priming outcomes can be significantly optimized by selecting cultivars with favorable underlying metabolic and immune backgrounds. To further refine these strategies, future work should focus on integrating metabolite profiling with the targeted evaluation of ARE and synergistic compounds beyond RA. This will enable tailored approaches for both genetically uniform, susceptible cultivars like Spunta and more resistant backgrounds such as Innovator. Ultimately, field studies under variable climate and pathogen pressures will be essential to validate the laboratory findings and assess the practical benefits of ARE priming for effective disease management.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAgehara S, Pride L, Gallardo M, Hernandez-Monterroza J (2020) A simple, inexpensive, and portable image-based technique for nondestructive leaf area measurements: HS1395, 11/2020. EDIS 2020. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.32473/EDIS-HS1395-2020\u003c/span\u003e\u003cspan address=\"10.32473/EDIS-HS1395-2020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAremu AO, Omogbene TO, Fadiji T, Lawal IO, Opara UL, Fawole OA (2024) Plants as an alternative to the use of chemicals for crop protection against biotic threats: trends and future perspectives. Eur J Plant Pathol 170:711\u0026ndash;766. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10658-024-02924-y\u003c/span\u003e\u003cspan address=\"10.1007/s10658-024-02924-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAr\u0026eacute;valo-Mar\u0026iacute;n DF, Brice\u0026ntilde;o-Robles DM, Mosquera T, Melgarejo LZ, Sarmiento F (2021). Jasmonic acid priming of potato uses hypersensitive response-dependent defense and delays necrotrophic phase change against Phytophthora infestans. Physiol Mol Plant Pathol 115: 101680. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.pmpp.2021.101680\u003c/span\u003e\u003cspan address=\"10.1016/j.pmpp.2021.101680\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArmstrong MR, Vossen J, Lim TY, et al (2019) Tracking disease resistance deployment in potato breeding by enrichment sequencing. Plant Biotechnol J 17:540\u0026ndash;549. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/pbi.12997\u003c/span\u003e\u003cspan address=\"10.1111/pbi.12997\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalint-Kurti P (2019) The plant hypersensitive response: concepts, control and consequences. Mol Plant Pathol 20:1163\u0026ndash;1178. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/mpp.12821\u003c/span\u003e\u003cspan address=\"10.1111/mpp.12821\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaulcombe DC, Chapman S, Santa Cruz S (1995) Jellyfish green fluorescent protein as a reporter for virus infections. Plant J 7:1045\u0026ndash;1053. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1365-313x.1995.07061045.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1365-313x.1995.07061045.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBradshaw JE (2022) A brief history of the impact of potato genetics on the breeding of tetraploid potato cultivars for tuber propagation. Potato Res 65:461\u0026ndash;501. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11540-021-09517-w\u003c/span\u003e\u003cspan address=\"10.1007/s11540-021-09517-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoban F, Ozer H, Yilmaz B, Lan Y (2025) Characterization of bioactive compounds in fenugreek genotypes in varying environments: diosgenin, trigonelline, and 4-hydroxyisoleucine. Front Plant Sci 16:1562931. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2025.1562931\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2025.1562931\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCohen Y, Gisi U (1994) Systemic translocation of 14C-dl-3-aminobutyric acid in tomato plants in relation to induced resistance against Phytophthora infestans. Physiol Mol Plant Pathol 45:441\u0026ndash;456. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0885-5765(05)80041-4\u003c/span\u003e\u003cspan address=\"10.1016/S0885-5765(05)80041-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCohen Y, Gisi U (2002) β-Aminobutyric Acid-induced resistance against plant pathogens. Plant Dis 86:448\u0026ndash;457. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1094/PDIS.2002.86.5.448\u003c/span\u003e\u003cspan address=\"10.1094/PDIS.2002.86.5.448\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoomber A, Saville A, Ristaino JB (2024) Evolution of Phytophthora infestans on its potato host since the Irish potato famine. Nat Commun 15:1\u0026ndash;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-024-50749-4\u003c/span\u003e\u003cspan address=\"10.1038/s41467-024-50749-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDefernez M, Gunning YM, Parr AJ, Shepherd LVT, Davies HV, Colquhoun IJ (2004) NMR and HPLC-UV profiling of potatoes with genetic modifications to metabolic pathways. J Agric Food Chem 52:6075\u0026ndash;6085. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jf049522e\u003c/span\u003e\u003cspan address=\"10.1021/jf049522e\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDess\u0026igrave; D, Fais G, Sarais G (2025) Nutritional and chemical characterization of red and purple potato peels: A polyphenol-rich by-product. Foods 14:1740. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/FOODS14101740\u003c/span\u003e\u003cspan address=\"10.3390/FOODS14101740\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDevaux A, Goffart JP, Petsakos A, et al (2019) Global food security, contributions from sustainable potato agri-food systems. In: The potato crop: Its agricultural, nutritional and social contribution to humankind, Springer I. pp 3\u0026ndash;35\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDieterle F, Ross A, Schlotterbeck G, Senn H (2006) Probabilistic quotient normalization as robust method to account for dilution of complex biological mixtures. Application in 1H NMR metabolomics. Anal Chem 78:4281\u0026ndash;4290. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ac051632c\u003c/span\u003e\u003cspan address=\"10.1021/ac051632c\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFavaro MA, Molina MC, Roeschlin RA, Gadea J, Gariglio N, Marano MR (2020) Different responses in mandarin cultivars uncover a role of cuticular waxes in the resistance to citrus canker. Phytopathology 110:1791\u0026ndash;1801. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1094/PHYTO-02-20-0053-R\u003c/span\u003e\u003cspan address=\"10.1094/PHYTO-02-20-0053-R\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFry WE (2016) Phytophthora infestans: New Tools (and old ones) lead to new understanding and precision management. Annu Rev Phytopathol 54:529\u0026ndash;547. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev-phyto-080615-095951\u003c/span\u003e\u003cspan address=\"10.1146/annurev-phyto-080615-095951\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGabriel J, Coca A, Plata G, Parlevliet JE (2007) Characterization of the resistance to \u003cem\u003ePhytophthora infestans\u003c/em\u003e in local potato cultivars in Bolivia. Euphytica 153:321\u0026ndash;328. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10681-006-9237-x\u003c/span\u003e\u003cspan address=\"10.1007/s10681-006-9237-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarcia L, Gerhardt N, Martin AP, Mart\u0026iacute;nez MF, Alemano S, Marano MR (2023) Tobacco necrosis virus A overcomes local cell death response in \u003cem\u003eNicotiana tabacum\u003c/em\u003e. Plant Pathol 72:154\u0026ndash;169. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/ppa.13629\u003c/span\u003e\u003cspan address=\"10.1111/ppa.13629\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarrett KA, Bebber DP, Etherton BA, et al (2022) Climate change effects on pathogen emergence: Artificial intelligence to translate big data for mitigation. Annu Rev Phytopathol 60:357\u0026ndash;378. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev-phyto-021021-042636\u003c/span\u003e\u003cspan address=\"10.1146/annurev-phyto-021021-042636\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhislain M, N\u0026uacute;\u0026ntilde;ez J, Del Rosario HM, et al (2009) Robust and highly informative microsatellite-based genetic identity kit for potato. Mol Breed 23:377\u0026ndash;388. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/S11032-008-9240-0\u003c/span\u003e\u003cspan address=\"10.1007/S11032-008-9240-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreenberg JT, Yao N (2004) The role of regulation of programmed cell death in plant-pathogen interactions. Cell Microbiol 6:201\u0026ndash;211. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1462-5822.2004.00361.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1462-5822.2004.00361.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrubb CD, Zipp BJ, Ludwig-M\u0026uuml;ller J, Masuno MN, Molinski TF, Abel S (2004) \u003cem\u003eArabidopsis\u003c/em\u003e glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis. Plant J 40:893\u0026ndash;908. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-313X.2004.02261.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-313X.2004.02261.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHalim VA, Eschen-Lippold L, Altmann S, Birschwilks M, Scheel D, Rosahl S (2007) Salicylic acid is important for basal defense of \u003cem\u003eSolanum tuberosum\u003c/em\u003e against \u003cem\u003ePhytophthora infestans\u003c/em\u003e. Mol Plant-Microbe Interact 20:1346\u0026ndash;1352. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1094/MPMI-20-11-1346\u003c/span\u003e\u003cspan address=\"10.1094/MPMI-20-11-1346\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e,\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe M, Ding NZ (2020) Plant unsaturated fatty acids: Multiple roles in stress response. Front Plant Sci 11:562785. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2020.562785\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2020.562785\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang S, Vleeshouwers VGAA, Visser RGF, Jacobsen E (2005) An accurate \u003cem\u003ein vitro\u003c/em\u003e assay for high-throughput disease testing of \u003cem\u003ePhytophthora infestans\u003c/em\u003e in potato. Plant Dis 89:1263\u0026ndash;1267. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1094/PD-89-1263\u003c/span\u003e\u003cspan address=\"10.1094/PD-89-1263\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJuarez ME (2024) Caracterizaci\u0026oacute;n poblacional, transcript\u0026oacute;mica y an\u0026aacute;lisis funcional de Phytophthora infestans en Argentina para la incorporaci\u0026oacute;n de resistencia al tiz\u0026oacute;n tard\u0026iacute;o de la papa. Dissertation (PhD), Universidad de Buenos Aires.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJuarez ME, Azcue J, Cano Mogrovejo ML, Lamour K, Bravo-Almonacid FF, Lucca AMF, Segretin ME (2024) Effector repertoire of an Argentinean Phytophthora infestans isolate and its relevance for late blight resistance deployment in potato. In: BSPP Plant Pathology 2024. Zenodo. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5281/zenodo.17087054\u003c/span\u003e\u003cspan address=\"10.5281/zenodo.17087054\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatahira R, Ashihara H (2009) Profiles of the biosynthesis and metabolism of pyridine nucleotides in potatoes (Solanum tuberosum L.). Planta 231:35\u0026ndash;45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00425-009-1023-2\u003c/span\u003e\u003cspan address=\"10.1007/s00425-009-1023-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKerchev P, van der Meer T, Sujeeth N, et al (2020) Molecular priming as an approach to induce tolerance against abiotic and oxidative stresses in crop plants. Biotechnol Adv 40: 107503. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biotechadv.2019.107503\u003c/span\u003e\u003cspan address=\"10.1016/j.biotechadv.2019.107503\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim HK, Choi YH, Verpoorte R (2010) NMR-based metabolomic analysis of plants. Nat Protoc 5:536\u0026ndash;549. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nprot.2009.237\u003c/span\u003e\u003cspan address=\"10.1038/nprot.2009.237\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLivak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-∆∆CT method. Methods 25:402\u0026ndash;408. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1006/meth.2001.1262\u003c/span\u003e\u003cspan address=\"10.1006/meth.2001.1262\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalcuit I, De Jong W, Baulcombe DC, Shields DC, Kavanagh TA (2000) Acquisition of multiple virulence/avirulence determinants by potato virus X (PVX) has occurred through convergent evolution rather than through recombination. Virus Genes 20:165\u0026ndash;172. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1023/A:1008178800366\u003c/span\u003e\u003cspan address=\"10.1023/A:1008178800366\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalcuit I, Marano MR, Kavanagh TA, De Jong W, Forsyth A, Baulcombe DC (1999) The 25-kDa movement protein of PVX elicits Nb-mediated hypersensitive cell death in potato. Mol Plant-Microbe Interact 12:536\u0026ndash;543. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1094/MPMI.1999.12.6.536\u003c/span\u003e\u003cspan address=\"10.1094/MPMI.1999.12.6.536\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarano MR, Malcuit I, De Jong W, Baulcombe DC (2002) High-resolution genetic map of Nb, a gene that confers hypersensitive resistance to potato virus X in Solanum tuberosum. Theor Appl Genet 105:192\u0026ndash;200. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00122-002-0962-9\u003c/span\u003e\u003cspan address=\"10.1007/s00122-002-0962-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartin AP, Mart\u0026iacute;nez MF, Chiesa MA, et al (2023) Priming crop plants with rosemary (\u003cem\u003eSalvia rosmarinus\u003c/em\u003e Spenn, syn \u003cem\u003eRosmarinus officinalis\u003c/em\u003e L.) extract triggers protective defense response against pathogens. Plant Physiol Biochem 197: 107644. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.plaphy.2023.107644\u003c/span\u003e\u003cspan address=\"10.1016/j.plaphy.2023.107644\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMondino MC, Grasso R, Balaban D, Ortiz MM (2021) Censo 2021 del Cintur\u0026oacute;n hort\u0026iacute;cola de Rosario. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.argentina.gob.ar/sites/default/files/2025/07/inta_oliveros_censo_horticola_2021.pdf\u003c/span\u003e\u003cspan address=\"https://www.argentina.gob.ar/sites/default/files/2025/07/inta_oliveros_censo_horticola_2021.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed 18 December 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMu\u0026ntilde;oz Hoyos L, Anisha WP, Meng C, et al (2024) Untargeted metabolomics reveals PTI-associated metabolites. Plant Cell Environ 47:1224\u0026ndash;1237. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/pce.14794\u003c/span\u003e\u003cspan address=\"10.1111/pce.14794\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNavarre DA, Brown CR, Sathuvalli VR (2019) Potato vitamins, minerals and phytonutrients from a plant biology perspective. Am J Potato Res 96:111\u0026ndash;126. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12230-018-09703-6\u003c/span\u003e\u003cspan address=\"10.1007/s12230-018-09703-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNavarre DA, Mayo D (2004) Differential characteristics of salicylic acid-mediated signaling in potato. Physiol Mol Plant Pathol 64:179\u0026ndash;188. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.pmpp.2004.09.001\u003c/span\u003e\u003cspan address=\"10.1016/j.pmpp.2004.09.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNicaise V (2014) Crop immunity against viruses: Outcomes and future challenges. Front Plant Sci 5:118474. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/FPLS.2014.00660/XML\u003c/span\u003e\u003cspan address=\"10.3389/FPLS.2014.00660/XML\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNyalugwe EP, Wilson CR, Coutts BA, Jones RAC (2012) Biological properties of potato virus x in potato: Effects of mixed infection with potato virus s and resistance phenotypes in cultivars from three continents. Plant Dis 96:43\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1094/PDIS-04-11-0305\u003c/span\u003e\u003cspan address=\"10.1094/PDIS-04-11-0305\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOsbourn AE (1996) Preformed antimicrobial compounds and plant defense against fungal attack. Plant Cell 8:1821\u0026ndash;1831. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1105/tpc.8.10.1821\u003c/span\u003e\u003cspan address=\"10.1105/tpc.8.10.1821\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePacheco R, Garc\u0026iacute;a-Marcos A, Barajas D, Marti\u0026aacute;\u0026ntilde;ez J, Tenllado F (2012) PVX-potyvirus synergistic infections differentially alter microRNA accumulation in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e. Virus Res 165:231\u0026ndash;235. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.virusres.2012.02.012\u003c/span\u003e\u003cspan address=\"10.1016/j.virusres.2012.02.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePang Z, Lu Y, Zhou G, et al (2024) MetaboAnalyst 6.0: towards a unified platform for metabolomics data processing, analysis and interpretation. Nucleic Acids Res 52:398\u0026ndash;406. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gkae253\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkae253\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePW T (1988) Use of uncontrolled freezing for liquid nitrogen storage of Phytophthora species. Plant Dis 72:680\u0026ndash;682\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR\u0026eacute;p\u0026aacute;s Z, Nagy R, Polg\u0026aacute;r ZG, Győri Z (2024) Study to determine the nutritional characteristics of potato varieties that are suitable for the application of the freeze-drying process. Potato Res 1\u0026ndash;23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11540-024-09827-9\u003c/span\u003e\u003cspan address=\"10.1007/s11540-024-09827-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalam U, Ullah S, Tang ZH, et al (2023) Plant metabolomics: An overview of the role of primary and secondary metabolites against different environmental stress factors. Life (Basel) 13:706. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/LIFE13030706\u003c/span\u003e\u003cspan address=\"10.3390/LIFE13030706\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalmer\u0026oacute;n A del M, Abreu AC, Trist\u0026aacute;n AI, et al (2025) Metabolic profiling of tomato plants infected with tomato brown rugose fruit virus: Insights into plant defense mechanisms and potential prebiotic interventions. ACS Agric Sci Technol 5:714\u0026ndash;724. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acsagscitech.4c00557\u003c/span\u003e\u003cspan address=\"10.1021/acsagscitech.4c00557\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSambrook, J.; Fritsch, E.F.; Maniatis T (1989) Molecular Cloning: A laboratory manual, 2nd edn. Cold Spring Harbor, NY, U.S.A.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS\u0026aacute;nchez G, Gerhardt N, Siciliano F, Vojnov A, Malcuit I, Marano MR (2010) 394\u0026ndash;405 Salicylic acid is involved in the Nb-mediated defense responses to potato virus X \u003cem\u003eSolanum tuberosum\u003c/em\u003e. Mol Plant-Microbe Interact 23:394\u0026ndash;405. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1094/MPMI-23-4-0394\u003c/span\u003e\u003cspan address=\"10.1094/MPMI-23-4-0394\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSavary S, Willocquet L, Pethybridge SJ, Esker P, McRoberts N, Nelson A (2019) The global burden of pathogens and pests on major food crops. Nat Ecol Evol 3:430\u0026ndash;439. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41559-018-0793-y\u003c/span\u003e\u003cspan address=\"10.1038/s41559-018-0793-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSyller J, Grupa A (2016) Antagonistic within-host interactions between plant viruses: Molecular basis and impact on viral and host fitness. Mol Plant Pathol 17:769\u0026ndash;782. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/MPP.12322\u003c/span\u003e\u003cspan address=\"10.1111/MPP.12322\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTagliotti ME, Deperi SI, Bedogni MC, et al (2018) Use of easy measurable phenotypic traits as a complementary approach to evaluate the population structure and diversity in a high heterozygous panel of tetraploid clones and cultivars. BMC Genet 19:1\u0026ndash;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12863-017-0556-9\u003c/span\u003e\u003cspan address=\"10.1186/s12863-017-0556-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTiwari JK, Buckseth T, Challam C, et al (2022a) CRISPR/Cas genome editing in potato: Current status and future perspectives. Front Genet 13:1\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fgene.2022.827808\u003c/span\u003e\u003cspan address=\"10.3389/fgene.2022.827808\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTiwari JK, Buckseth T, Zinta R, et al (2022b) Germplasm, breeding, and genomics in potato improvement of biotic and abiotic stresses tolerance. Front Plant Sci 13: 805671. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2022.805671\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2022.805671\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Wersch S, Tian L, Hoy R, Li X (2020) Plant NLRs: The whistleblowers of plant immunity. Plant commun.1:100016. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.xplc.2019.100016\u003c/span\u003e\u003cspan address=\"10.1016/j.xplc.2019.100016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan West P, De Jong AJ, Judelson HS, Emons, Anne MC, Govers F (1998) The ipiO gene of \u003cem\u003ePhytophthora infestans\u003c/em\u003e is highly expressed in invading hyphae during infection. Fungal Genet Biol 23:126\u0026ndash;138. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1006/fgbi.1998.1036\u003c/span\u003e\u003cspan address=\"10.1006/fgbi.1998.1036\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerchot J (2022) Potato virus X: A global potato-infecting virus and type member of the Potexvirus genus. Mol Plant Pathol 23:315\u0026ndash;320. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/mpp.13163\u003c/span\u003e\u003cspan address=\"10.1111/mpp.13163\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVeselkov KA, Lindon JC, Ebbels TMD, et al (2009) Recursive segment-wise peak alignment of biological 1H NMR spectra for improved metabolic biomarker recovery. Anal Chem 81:56\u0026ndash;66. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ac8011544\u003c/span\u003e\u003cspan address=\"10.1021/ac8011544\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVleeshouwers VGAA, Van Dooijeweert W, Govers F, Kamoun S, Colon LT (2000) Does basal PR gene expression in \u003cem\u003eSolanum\u003c/em\u003e species contribute to non-specific resistance to \u003cem\u003ePhytophthora infestans\u003c/em\u003e? Physiol Mol Plant Pathol 57:35\u0026ndash;42. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1006/pmpp.2000.0278\u003c/span\u003e\u003cspan address=\"10.1006/pmpp.2000.0278\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVollmer R, Espirilla J, Villagaray R, et al (2021) Cryopreservation of potato shoot tips for long-term storage. In: Methods in molecular biology. Humana Press Inc., pp 21\u0026ndash;54\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWishart DS, Jewison T, Guo AC, et al (2013) HMDB 3.0-The human metabolome database in 2013. Nucleic Acids Res 41: D801-807. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gks1065\u003c/span\u003e\u003cspan address=\"10.1093/nar/gks1065\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu EJ, Wang YP, Yang LN, Zhao MZ, Zhan J (2022) Elevating air temperature may enhance future epidemic risk of the plant pathogen \u003cem\u003ePhytophthora infestans\u003c/em\u003e. J fungi (Basel) 8:808. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/JOF8080808\u003c/span\u003e\u003cspan address=\"10.3390/JOF8080808\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu XQ, Niu HQ, Liu C, Wang HL, Yin W, Xia X (2024) PTI-ETI synergistic signal mechanisms in plant immunity. Plant biotechnol J 22:2113\u0026ndash;2128. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/pbi.14332\u003c/span\u003e\u003cspan address=\"10.1111/pbi.14332\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang P, Jackson E, Li X, Zhang Y (2025) Salicylic acid and jasmonic acid in plant immunity. Hortic res 12:uhaf082. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/hr/uhaf082\u003c/span\u003e\u003cspan address=\"10.1093/hr/uhaf082\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang S, Huang A, Lv X, et al (2023) Anti-oomycete effect and mechanism of salicylic acid on Phytophthora infestans. J Agric Food Chem 71:20613\u0026ndash;20624. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jafc.3c05748\u003c/span\u003e\u003cspan address=\"10.1021/acs.jafc.3c05748\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu C, Wu S, Sun T, et al (2021) Rosmarinic acid delays tomato fruit ripening by regulating ripening-associated traits. Antioxidants 10:1821. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/antiox10111821\u003c/span\u003e\u003cspan address=\"10.3390/antiox10111821\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Plant immunity, Rosemary, NMR metabolomics, Biocontrol, Priming","lastPublishedDoi":"10.21203/rs.3.rs-8399039/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8399039/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePotato (\u003cem\u003eSolanum tuberosum\u003c/em\u003e L.) is a major global food crop increasingly threatened by pathogens such as Potato virus X (PVX) and \u003cem\u003ePhytophthora infestans\u003c/em\u003e. Priming with plant extracts, including rosemary aqueous extract (ARE), provides a sustainable strategy to enhance crop immunity. Here, constitutive and ARE-induced defense responses were analyzed across four commercial cultivars: Innovator, Kennebec, Spunta, and Frital-INTA. \u003csup\u003e1\u003c/sup\u003eH NMR metabolomic profiling combined with defense gene expression analysis under non-infected conditions revealed cultivar-specific signatures, suggesting that basal metabolism and genetic background influence pathogen susceptibility and can be selectively tuned by ARE application. Subsequent infection assays with PVX and \u003cem\u003eP. infestans\u003c/em\u003e validated these differential responses, identifying Innovator as more resistant and Spunta as more susceptible. Crucially, ARE pre-treatment significantly enhanced defense responses, particularly in susceptible cultivars. This priming effect resulted in a marked reduction in PVX accumulation and a decrease in \u003cem\u003eP. infestans\u003c/em\u003e lesion size. These findings extend the established efficacy and sustainability of ARE to potato cultivation, demonstrating its capacity to act as a potent priming agent. Specifically, our results show that ARE reinforces potato immunity by integrating and amplifying both constitutive and inducible defense mechanisms, further highlighting its position as a versatile bioprotective tool for crop disease management\u003c/p\u003e","manuscriptTitle":"Rosemary extract primes cultivar-dependent defense responses in potato against pathogen attack","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-09 14:14:46","doi":"10.21203/rs.3.rs-8399039/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-18T00:53:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-14T19:51:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-13T14:05:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"177746045309913662569624833531995974637","date":"2026-01-07T16:32:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"227334996141388313820752966610166478708","date":"2026-01-07T16:30:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"291181510153319932882633264020530668207","date":"2026-01-07T15:24:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-07T14:54:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-20T11:05:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-20T11:04:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2025-12-18T21:42:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5f8fc746-c96e-4549-a1f2-33b4dc493b8d","owner":[],"postedDate":"January 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:23:25+00:00","versionOfRecord":{"articleIdentity":"rs-8399039","link":"https://doi.org/10.1007/s00299-026-03787-9","journal":{"identity":"plant-cell-reports","isVorOnly":false,"title":"Plant Cell Reports"},"publishedOn":"2026-03-28 16:11:08","publishedOnDateReadable":"March 28th, 2026"},"versionCreatedAt":"2026-01-09 14:14:46","video":"","vorDoi":"10.1007/s00299-026-03787-9","vorDoiUrl":"https://doi.org/10.1007/s00299-026-03787-9","workflowStages":[]},"version":"v1","identity":"rs-8399039","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8399039","identity":"rs-8399039","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}