About 10,000 years of grapevine evolution and domestication in almost 90 countries (Villano and Aversano, 2020) provided a high number of genotypes possessing various morphological and genetic traits (This et al., 2006). However, the selection process carried out by humans shaped the gene pool of today’s varieties with valuable traits in the sense of yield, chemical composition, berry and bunch size, phenology, hermaphrodite flowers etc., while resistance to main pathogens was unintentionally neglected (Grassi and Arroyo-Garcia, 2020). Nowadays, it is more than ever necessary to produce enough food for the growing human population in a way that achieves a minimal footprint on the environment. That is where breeding programs play a major role and aim to bring about high-quality cultivars that can cope with the difficulties of main diseases. When it comes to the grapevine, downy mildew is one of the most destructive diseases. The causal agent of this disease is Plasmopara viticola [(Berk. and Curt.) Berl. and de Toni], which is one of the most damaging pathogens affecting grapevine production in all viticultural regions worldwide (Armijo et al., 2016). Diseases like mildews are allochthonous in Europe and therefore, grapevine (Vitis vinifera L.) varieties are susceptible to them unlike the American species [e.g., Vitis riparia Michx, Vitis labrusca L., Vitis rupestris Scheele, Muscadinia rotundifolia Small (previously Vitis rotundifolia Michaux)] which developed resilience coevolving on the same geographical area (Jürges et al., 2009; Gessler et al., 2011). A considerable level of resistance to downy mildew is observed in the Asian species, such as Vitis amurensis Rupr, which coevolved with the species of the pathogen closely related to P. viticola, i.e., Plasmopara cissi Vienn.-Bourg and Plasmopara amurensis Prots (Dick, 2002).

Plasmopara viticola is an obligate biotrophic oomycete meaning that it feeds on the living tissue, through haustoria in order to invade the host cell and obtain plant metabolites (Glazebrook, 2005). Its sporangia have lemon-shaped coenocytic cells that contain four to eight nuclei (Riemann et al., 2002). During the grapevine growing season, when conditions for downy mildew development are favorable, symptoms of infection appear on the green tissues (i.e., leaves, tendrils, inflorescences, shoots, and green berries), always starting with the young leaves. For that reason, in vitro experiments on the leaves are often used as an indicator of a variety’s susceptibility to P. viticola (Jürges et al., 2009; Bove et al., 2019). Visible adaxial symptoms on the leaves, called oil spots, are reported to usually precede the abaxial whitish sporulation (Gessler et al., 2011). When fungicides are not applied during favorable weather conditions for downy mildew development, it can devastate almost the whole yield in one season and weaken the young shoots, causing a considerable economic loss (Buonassisi et al., 2017). Yet, fungicides, both in the organic farming as copper fungicides and in the Integrated Pest Management even with other active substances, act harmfully to the environment, and animal and human health (Wilson and Tisdell, 2001); thus breeding programs aim to produce genotypes with efficient and durable resistance to main diseases, such as mildews and gray mold (Merdinoglu et al., 2018).

The mode of plant-pathogen interaction begins when the initial contact is established between infective propagules (P. viticola zoospores) and the plant tissue surfaces (e.g., leaf lamina). To prevent the diseases caused by pathogens, plants use sophisticated defense mechanisms that can be either constitutive or inducible defenses (Muganu and Paolocci, 2013). While the constitutive defense is referred to as a preexisting and continuous resistance (Kono and Shimizu, 2020), the induced defense is triggered by a pathogen attack and recognition and includes the perception of plant tissue signals resulting from pathogen infection (Muganu and Paolocci, 2013). Constitutive defense includes preformed physical barriers present on the plant surface (leaf hairs, wax layers, rigid cell walls, and the number and the activity of stomata) or chemical compounds, such as antimicrobial secondary metabolites (Lattanzio et al., 2006). These compounds are called phytoanticipins, which are defined as compounds that are present in plants before being challenged by microorganisms or are produced after infection solely from preexisting constituents (VanEtten et al., 1994). It has already been emphasized that increasing knowledge about constitutive phytoanticipins, such as leaf polyphenols could be pivotal to explaining the different levels of susceptibility to pathogens displayed by V. vinifera genotypes (Kedrina-Okutan et al., 2018). On the other hand, there are compounds that are produced by plants as a response to biotic and abiotic stresses called phytoalexins (Jeandet, 2015). Upon P. viticola infection of grapevine leaves, the synthesis of stilbenes is usually induced, among which resveratrol is the most common compound. It can reduce the germination of spores, which proves its strong antimicrobial activity against P. viticola (Dercs and Creasy, 1989). Scarce information is available suggesting that specific profiles exist at the transcriptome and metabolome level that can discriminate susceptible and resistant cultivars before being infected with P. viticola (Figueiredo et al., 2008).

Up to date, numerous studies have been published considering the composition and content of secondary metabolites, namely polyphenolic and volatile organic compounds, in grapevine leaves before and after P. viticola infection aiming to elucidate which compounds are specifically responsible for a certain level of tolerance to this microorganism among different species and varieties (Figueiredo et al., 2008, 2015; Batovska et al., 2009; Chitarrini et al., 2017; Eisenmann et al., 2019; Ciubotaru et al., 2021; Ricciardi et al., 2021). On the other hand, studies focused on the differences among V. vinifera varieties with different levels of resistance and their metabolomic discrimination either before or after inoculation with P. viticola are deficient. As a part of plants’ secondary metabolism, polyphenolic compounds and phenolic acids are not directly involved in their growth, development, and reproduction; yet they eminently participate and influence these processes. They are located in the epidermis of the leaves (cell vacuoles), cuticle, and epicuticular wax—predominantly, in the outer layers of the leaves. This epidermis/cuticle skin forms the first mechanical barrier to invading pathogens by repelling fungal spores due to its self-cleaning surface (Keller, 2020). Moreover, one of the most important roles of polyphenolics is the defense reaction due to their antifungal and antibacterial properties (Lattanzio et al., 2006). Polyphenol accumulation and profiles are influenced by seasonal climatic conditions, biotic and abiotic stressors, soil, cultural practices, and genetics (Kedrina-Okutan et al., 2018). In stressed plants, the level of reactive oxygen species (ROS) is surpassed over the antioxidant compounds. Stressors can induce the activation of the defense mechanism, which increases the biosynthesis of many phenolic compounds (Bouderias et al., 2020).

Until recently, it was thought that genetic variability among V. vinifera germplasm is too scarce in a sense of resistance to main fungal diseases. However, in some V. vinifera varieties, such as Kishmish vatkana, Dzandzal kara (Coleman et al., 2009), and Mgaloblishvili (Sargolzaei et al., 2020), resistance genes have been identified. From this kind of research, it can be concluded that varieties of local importance are possible sources of desirable features that can be useful in the upcoming changing climate as some of them are able to cope with abiotic (drought, salinity, iron chlorosis) and biotic stresses (Sargolzaei et al., 2021).

Croatia is a country with a long tradition in grapevine cultivation with many climatically diverse regions that provided to develop a high number of native grapevine varieties. The introduction of phylloxera and mildews at the end of the nineteenth century gradually caused the erosion of this preceding germplasm. Thus, today’s native collection counts slightly more than a hundred varieties (Maletić et al., 2015a; Žulj Mihaljević et al., 2020). Due to centuries-old grapevine cultivation and their adjustment to disparate environmental conditions, there is a presumption that diverse responses to diseases exist among the Croatian native varieties. These differences were recently confirmed on a series of studies applying field research, the leaf disc bioassay (OIV, 2009), and by measuring the chlorophyll fluorescence and multispectral imaging traits (Štambuk et al., 2021). In the present study, this research is extended to the metabolomic approach aiming (1) to examine the differences in the content of the polyphenols during the early stage of infection of V. vinifera varieties with different degree of resistance to P. viticola and (2) to assess the existence of a correlation between the polyphenolic profiles of 14 Croatian native V. vinifera genotypes, and their belonging to different classes of resistance to P. viticola according to the classification of the International Organization of Vine and Wine (OIV). To the best of our knowledge, up to now, there has been no research that included such a high number (15) of V. vinifera varieties considering their constitutive and induced leaf polyphenolic profiles regarding the level of susceptibility to P. viticola. Therefore, this study provides an invaluable source of information that could be used for screening other vinifera varieties with no defined level of susceptibility to this pathogen and to improve the success of grapevine breeding programs toward downy mildew resistance.

Among the phenolic compounds, 10 flavan-3-ols, nine flavonol glycosides, eight hydroxycinnamic acids, four hydroxybenzoic acids, and two stilbenes were detected (Supplementary Table 1). The most abundant class of phenolic compounds were hydroxycinnamic acids with an average value of 25.19 g/kg among which caftaric acid (4.99 g/kg) contributed the most. Hydroxycinnamic acids were followed by flavonol glycosides (20.2 g/kg), flavan-3-ols (5.4 g/kg), hydroxybenzoic acids (227.32 mg/kg), and stilbenes (151.72 mg/kg). As far as individual compounds are concerned, the highest amount was detected for quercetin-3-O-glucoside (26.39 g/kg) in the samples representing the inoculated leaves of the OIV class 3 at 48 hpi (T₂) (Supplementary Table 1). Correlation between the content of the phenolic compounds and the terms of sampling was significant only in the case of resveratrol-3-O-glucoside and total stilbenes in both the inoculated and non-inoculated samples (Supplementary Table 2). There was no significant correlation found between OIV resistance classes and the density of prostrate hairs between the main veins on the lower side of the young leaves.

Studies considering metabolomic changes of the grapevines and profiling regarding the different levels of susceptibility to P. viticola have been intriguing for more than a decade and this trend does not seem to fade. For this purpose, HPLC proved to be a reliable and scrutinized analytical technique by which it is possible to quantify phenolic acids and polyphenolic compounds (Tomaz and Maslov, 2016). As possible progress of breeding programs that are oriented toward improved resistance, V. vinifera varieties are in the spotlight to research and use as progenitors since most of them do not contain undesirable viticultural and oenological features like American species (Toepfer et al., 2011).

Plant metabolites can be either included in the primary metabolism, such as lipid compounds, amino acids, and sugars, or secondary metabolism, such as phenolic compounds arising biogenetically from the shikimate-phenylpropanoids-flavonoids pathways (Lattanzio et al., 2006). Some of the important physiological roles of phenolic compounds are the formation of the cell wall polymers, lignin (Paone et al., 2020) and suberin (Bernards and Razem, 2001), floral and fruit pigment synthesis (Tanaka et al., 2008), ultraviolet sunscreen protection (Cefali et al., 2016), formation of flavor compounds (Kielhorn and Thorngate, 1999), and defense against pathogens (Zaynab et al., 2018).

Gómez-Zeledón and Kaiser (2016); Buonassisi et al. (2018), and Vezzulli et al. (2018) have proposed OIV descriptor 452-1 (OIV, 2009), and therefore it was also applied in the research aiming to distinguish groups of resistance among Croatian native grapevine varieties as this method is reliable with included control genotypes (Štambuk et al., 2021). Nonetheless, for comprehensive research purposes, in the present work, the screening is extended and dedicated to the analysis of secondary metabolites, namely polyphenolic compounds and phenolic acids aiming to define their possible relation with differences in the resistance level among them.

The most abundant group of polyphenolic compounds detected in the leaves of genotypes included in this study were hydroxycinnamic acids (25.19 g/kg DW) followed by flavonol glycosides, whose content was slightly lower (20.2 g/kg DW). Flavonol glycosides have photoprotective roles by filtering the UV-B light while allowing to pass photosynthetically active visible light (Agati et al., 2013) together with an antioxidant function during plant response to environmental stress (Hernández et al., 2009). Quercetin-3-O-glucoside was the most abundant flavonol in the research performed by Anđelković et al. (2015) in the grapevine leaf extracts from Vranac and Merlot (V. vinifera). Previous studies confirmed the higher susceptibility of shaded leaves grown in the greenhouse that contains less flavonols (Agati et al., 2008; Latouche et al., 2013) thus supporting the results of the present study where hydroxycinnamic acids are the most abundant polyphenolic group, probably due to reduced ultraviolet radiation conditions in the greenhouse. According to Meyer et al. (2021), supplemental UV-B light has a positive effect on disease resistance in many plant-pathogen combinations, mainly through the induction of the production of specialized metabolites like flavonoids.

No significant differences were found between the control and the inoculated leaves regardless of the OIV class and the term of sampling, with the exception of stilbenes and resveratrol-3-O-glucoside specifically. That is in accordance with the previous study where non-destructive optical methods were used (Latouche et al., 2013) for monitoring flavonols, hydroxycinnamic acids, and stilbenes throughout 6 days upon P. viticola inoculation in the Cabernet Sauvignon leaves grown in the greenhouse. Resveratrol-3-O-glucoside also showed a significant positive correlation in terms of sampling both in the case of inoculated and non-inoculated samples. Stilbenes are the most important class of phytoalexins in the Vitaceae family, which are dynamically accumulated in response to various abiotic and biotic stresses, including pathogen attacks (Ciaffi et al., 2019). Thus, their ascending content in the non-inoculated leaves could be explained by picking them as well as changing the environment from the greenhouse to the laboratory conditions. Moreover, inoculation with P. viticola suspension caused additional stress and thus in these leaves, the contents of resveratrol-3-O-glucoside and total stilbenes were even higher throughout the experiment. Previous studies have shown that the accumulation of higher stilbenes is usually associated with the response of the resistant genotypes to P. viticola infection (Boso et al., 2012; Chitarrini et al., 2017). In our study, huge variability was detected among the genotypes belonging to different resistance classes confirming that the accumulation of stilbene resveratrol-3-O-glucoside is related to infection. However, the obtained results also suggest that increased content of resveratrol-3-O-glucoside is not sufficient to achieve a high level of resistance to P. viticola. In response to the presence of P. viticola, stilbenes are synthesized in grapevine leaves (Chalal et al., 2014). They possess antimicrobial activity that may be strong enough to inhibit the infection in resistant genotypes (Chong et al., 2009), which is not accurate for susceptible genotypes. Based on the previous studies, the content of stilbenes increases in accordance with P. viticola development in susceptible varieties suggesting that the accumulation of stilbenes can be used as an indicator of P. viticola infection (Naidenov et al., 2010; Latouche et al., 2013).

In the present work, three susceptible OIV classes (1, 3, and 5) are distinguished by each group characteristic of polyphenolic compounds provided by PCA. More specifically, the most susceptible OIV class 1 was separated from two other groups by being abundant in caffeic and vanillic acid, which are hydroxycinnamic and hydroxybenzoic acids, respectively. Caffeic acid has also been found in high amounts in susceptible V. vinifera varieties (Riesling Weiss, Pinot Noir, Cabernet Sauvignon, and Trincadeira) (Maia et al., 2020). On the contrary, caffeic acid has been previously related to constitutive resistance in the partially resistant cultivar Regent (Figueiredo et al., 2008). This compound participates in enzymatic oxidative mechanisms in response to the pathogenic infection of the grapevine (Mattivi et al., 2011). Among flavan-3-ols, the only discriminator was epigallocatechin-gallate known for its high antioxidant capacity (Kedrina-Okutan et al., 2018).

Flavan-3-ols, i.e., catechin and epicatechin, were more abundant in the presented OIV class 3. A previous study (Maia et al., 2020) hypothesizes that higher levels of catechin/epicatechin and over-expression of LAR2 gene (involved in the conversion of leucocyanidin into catechin and epicatechin) may be putative biomarkers of susceptibility. Catechin, together with other phenolic compounds, possesses antioxidant properties and has been previously determined as a part of the grapevine defense mechanism (Kortekamp, 2006). However, there is a presumption that catechin can be degraded by different fungi, used as a carbon source for growth, and finally used for establishing a successful infection (Maia et al., 2020), but the precise potential of P. viticola in the degradation of this compound is not investigated. Epicatechin has been proposed as a biomarker of resistance in a study by Ciubotaru et al. (2021) due to its higher content in the genotype, BC4 possessing resistant locus Rpv1. Phenolic acids, namely fertaric, coutaric, and gallic acid, have also contributed to the discrimination. Nevertheless, Ali et al. (2012) identified fertaric acid in the partially resistant cultivar Regent.

Quercetin-3-O-glucoside was a discriminative compound that was more abundant in the OIV class 5, which is in accordance with a previous study (Maia et al., 2020) where the same flavonol glycoside together with several others was found in higher concentrations in the resistant/partially resistant genotypes. Another flavonol, namely kaempferol-3-O-rutinoside that distinguished this class, was also detected previously in the partially resistant cultivar, Bianca at 12 hpi (Chitarrini et al., 2017). Furthermore, quercetin-3-O-glucoside and caftaric acid were found at higher concentrations and therefore were responsible for distinguishing Regent from Trincadeira (Ali et al., 2012). Latouche et al. (2013) observed that constitutive higher content of flavonols slowed down the accumulation of stilbenes in the grapevine leaves, and thus the phytoalexin-mediated response of leaves to P. viticola was delayed, suggesting that constitutive higher amounts of flavonols could confine the spreading of the pathogen. Trans-caftaric acid was the most abundant phenolic acid in the leaf extracts of Vranac and Merlot, with lower content in infected ones ( Anđelković et al., 2015). Ferulic and p-coumaric acids were also discriminative for the OIV 5. The highest contents of these acids were previously found in the interspecies hybrid Petra, with 12.5% of Vitis amurensis and 87.5% of Vitis vinifera in its genetic background, among other pure V. vinifera varieties (Pantelić et al., 2017). Petra is known for high cold hardiness and reduced susceptibility to P. viticola and Botrytis cinerea (Cindric et al., 2003).

Apart from constitutive and induced chemical compounds that provide a certain level of tolerance to parasitic microorganisms, resistance to P. viticola can be associated with the synthesis of physical barriers, such as callose and lignin appositions (Toffolatti et al., 2012). Moreover, hydrophobic trichomes on the abaxial leaf sides reduce the retention or repel water drops, thus preventing the encystment of P. viticola zoospores (Kono and Shimizu, 2020), a step that is essential for the pathogen development inside a leaf tissue and further fructification (Rossi and Caffi, 2007, 2012). This morphological feature is an example of passive resistance, whereas active responses involve hypersensitivity and synthesis of specific secondary metabolites (Buonassisi et al., 2017). The morphological characteristic of Croatian native varieties, i.e., Teran and Ranfol, is abaxial leaf sides covered by extremely dense hydrophobic and moderately dense trichomes, respectively, that certainly obstruct P. viticola sporangia to reach the epidermis and stoma at the leaf bottom. On the other hand, the leaves of Malvazija istarska are glabrous; yet they are firm and robust (Maul et al., 2012; Maletić et al., 2015a) whose possibly thick cuticle protect them from plant pathogens (Serrano et al., 2014). There are varieties with a relatively high density level of trichomes within the classes of resistance, such as Belina starohrvatska, Moslavac, and Plavac mali in class 1, Plavina in class 3, and Ranfol and Teran in class 5. Opposed to this, in the case of resistant genotypes (class 7 and 9), low density levels of the trichomes are present. Subsequently, no correlation between the density of the trichomes and resistance to P. viticola was determined suggesting that this feature does not have a major effect on the resistance level of specific genotypes, in contrast to some previous studies (Kortekamp and Zyprian, 1999; Kono and Shimizu, 2020).

Solaris, one of the control varieties used in this research, proved its high yet not complete resistance to P. viticola (OIV 452 ≈ 7) in previous studies (Vezzulli et al., 2018; Ciubotaru et al., 2021) since this variety contains two resistance genes (Rpv3-3 and Rpv10) (Vezzulli et al., 2019; Possamai et al., 2020). Such a pyramided resistance provides a higher level of resistance generally expressed as a more stable and durable feature (Merdinoglu et al., 2018). However, it was found that its response to P. viticola infection is isolate-specific and highly variable (Heyman et al., 2021). Due to its genetic background based on V. vinifera [Merzling × (Zarya Severa × Muscat Ottonel)] (Pezet et al., 2004), it reacted more similarly to V. vinifera varieties, when compared to V. riparia upon P. viticola inoculation. This is comparable with previous research where Regent’s (on which backcrosses were made with V. vinifera) metabolic profile clustered together with V. vinifera varieties (Maia et al., 2020). In Bianca, which has an Rpv3 locus in its genome, the content of the secondary metabolites increased at later stages after the infection (96 hpi). These were phenylpropanoids, flavonols, and stilbenes, whereas the earliest modifications included primary metabolites, i.e., lipids, amino acids, acids, and sugars at 24–48 hpi (Chitarrini et al., 2017).

Vitis riparia is an indigenous species to North America where it evolved with fungi/oomycete, E. necator and P. viticola, and subsequently developed resistance to mildew diseases (OIV 452 = 9). Low or no sporulation values were associated with this genotype in the previous studies (Boso et al., 2012; Bhattarai et al., 2021). Thus, it has been effectively used in breeding programs for resistance introgression (Toepfer et al., 2011). Upon P. viticola infection, this genotype produced the highest content of resveratrol-3-O-glucoside, piceatannol, and total stilbenes which have been observed previously (Boso et al., 2012) due to the fast constitutive expression of the stilbene synthase genes as well as the extent of their transcriptional activation following P. viticola inoculation (Ciaffi et al., 2019). Stilbenes are toxic to phytopathogenic fungi and may contribute to disease resistance as phytoalexins (Ribera and Zuñiga, 2012). Although stilbenes were also identified in susceptible genotypes, they contributed to the differentiation of the OIV class 1; their importance is much greater in discriminating the resistant genotype. Along with stilbenes, epigallocatechin-gallate and kaempferol-3-O-rutinoside discriminated the OIV class 9 from all other OIV classes. Kedrina-Okutan et al. (2018) stated that V. riparia leaves constitutively contain a higher amount of total polyphenols, total flavonols, and total phenolic acids compared to V. rupestris, which could explain its specifically high resistance to P. viticola, as the flavonols limit this pathogen development (Ali et al., 2012). Comparing metabolic compositions associated with disease susceptibility of different Vitis species and V. vinifera varieties, V. riparia clustered together with V. labrusca, V. candicans, V. vinifera subsp. sylvestris, and V. rotundifolia, whereas the Regent was closer to V. vinifera varieties, such as Riesling Weiss and Pinot Noir (Maia et al., 2020), confirming the results of the present study. The susceptible control variety, Chardonnay was previously included in a study (Toffolatti et al., 2012) where the changes of antifungal compounds upon P. viticola infection are described and flavonoids showed no specific reaction to the presence of this pathogen. In our study, the polyphenolic profile of Chardonnay was similar to most of the other genotypes belonging to OIV class 3, although some compounds (i.e., protocatechuic acid, gallocatechin, procyanidins B1, B3, and B4) were higher than in the case of other genotypes from this class. Chardonnay and the native variety, Kraljevina were specific for the highest content of gallic acid.

There were no previous studies on the polyphenolic composition of the leaves of Croatian native varieties used in this study and our previous study (Štambuk et al., 2021) was the first one on the susceptibility of Croatian grapevine germplasm. Results from both of these studies, confirm the high level of variability within the Croatian native varieties previously defined for other important characteristics (Maletić et al., 2015b).

This work has demonstrated the importance of secondary metabolites in grapevine defense responses against P. viticola with particular emphasis on Croatian native varieties. The research is based on a detailed analysis of phenolic compounds responsible for the discrimination of varieties among the OIV classes of resistance. The performed polyphenolic analysis confirmed and fulfilled the previous studies suggesting that constitutive polyphenolic profile contributes to the separation of susceptible OIV classes (1, 3, and 5) into three groups. The high variability in the content of resveratrol-3-O-glucoside and total stilbenes was determined, and discrimination among non-infected and infected samples was detected. However, the content of this compound did not show a clear difference between the resistant and susceptible genotypes. The content of piceatannol and total stilbenes discriminated completely resistant OIV class 9 (V. riparia) and the remaining OIV classes, thus confirming their strong antimicrobial properties. Considering the polyphenolic profiles of V. vinifera varieties, mostly flavonol glycosides were found to be responsible for lower susceptibility. Multivariate analysis shows complex relations among phenolic profiles and resistance levels suggesting that the preinfectional phenolic profile of leaves could be a determinant for different susceptibility to P. viticola.

Less susceptible grapevine varieties that belong to OIV class 5 (Malvazija istarska, Ranfol, Teran) could be interesting to use in breeding programs aiming to produce high-quality genotypes resistant to main fungal diseases. A further intention is directed toward analyzing constitutive and induced volatile organic compounds since their profile should distinguish grapevine classes of susceptibility to P. viticola likewise, whereas potential early metabolomic changes should elucidate additional bioactive molecules.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

PŠ, JK, and IT contributed to the conceptualization of the study. PŠ and IT contributed to the methodology. PŠ, IŠ, and DP conducted the formal analysis. PŠ and IŠ performed the investigations. DP performed data curation. PŠ wrote the original draft preparation. IŠ, DP, JK, EM, and IT contributed to the writing, reviewing, and editing. JK and IT supervised the work. EM contributed to funding acquisition. All authors contributed to the manuscript revision, read, and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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