Stearic Acid in Grapevine Cuticular Wax Acts as a Chemical Stimulator of Erysiphe necator Conidial Germination

Abstract

Cuticular wax metabolites on leaf surfaces function as chemical interfaces that modulate the pre-penetration development of phytopathogenic fungi. However, the specific monomeric constituents in grapevine leaf wax that regulate conidial germination and host recognition by grapevine powdery mildew caused by Erysiphe necator remain elusive. Here, we compared leaf cuticular wax characteristics between four susceptible Vitis vinifera cultivars and three resistant Chinese wild Vitis accessions. Scanning electron microscopy coupled with gravimetric analysis revealed that susceptible V. vinifera cultivars exhibited lower wax loads and lamellar crystals, whereas resistant Chinese wild Vitis accessions displayed higher wax accumulation with granular or blocky crystals. Gas chromatography–mass spectrometry profiling further demonstrated divergent compositional patterns: primary alcohols predominated among susceptible V. vinifera cultivars, while aldehydes and alkanes were enriched in resistant Chinese wild Vitis accessions. Paradoxically, in vitro conidial germination assays showed that wax extracts from all grapevines significantly promoted En. NAFU1 conidial germination, indicating that resistance in Chinese wild Vitis accessions is independent of direct wax-mediated inhibition of spore germination. Bioassays of representative monomers revealed that n-triacontane suppressed germination at high concentrations, whereas n-dotriacontanol and stearic acid stimulated it. Collectively, these findings demonstrate that specific cuticular wax monomers exert distinct biological effects on En. NAFU1 conidial germination. Given the ubiquitous presence and concentration-dependent stimulatory activity of stearic acid, we hypothesize that this monomer serves as a conserved chemical cue enabling initial host recognition by En. NAFU1, whereas the differential susceptibility among grapevine genotypes is governed by downstream physical and induced defense mechanisms rather than by variation in stearic acid levels.

1. Introduction

Cuticular waxes constitute a hydrophobic protective barrier on aerial plant organs, which consist of intracuticular waxes embedded within the cutin matrix and epicuticular waxes coating the external surface [1,2]. Chemically, these waxes are dominated by C20–C34 long-chain fatty acids and their aliphatic derivatives, including alkanes, alcohols, aldehydes, ketones, and esters, whereas certain taxa additionally contain branched alkanes, alkenes, triterpenoids, sterols, flavonoids, and tocopherols [3,4,5]. Through molecular self-assembly, these hydrophobic constituents generate distinctive crystalline microstructures, including platelets, tubules, rods, granules, and amorphous films, whose supramolecular architecture is strongly governed by chemical composition. Specifically, primary alcohols characteristically form platelet crystals, whereas secondary alcohols and β-diketones preferentially form hollow tubular configurations [6,7]. The supramolecular architecture of these wax crystals confers superhydrophobicity and self-cleaning properties to the phylloplane, thereby minimizing cuticular water loss, attenuating ultraviolet (UV) radiation, and restricting pathogen ingress [8,9].

The morphology of epidermal wax crystals correlates with pathogen resistance, where compact wax deposits and distinct crystalline morphologies provide a physical barrier to conidial adhesion and pathogen ingress [10,11]. In addition to their physical barrier function, cuticular wax constituents operate as biochemical cues that affect the germination and differentiation of several plant pathogenic fungi, including the barley powdery mildew (Blumeria graminis) [12,13], the Arabidopsis powdery mildew (Golovinomyces orontii) [14], the citrus green mold (Penicillium digitatum) [15], the castor gray mold (Amphobotrys ricini) [16], and the pear black rot (Alternaria alternata) [17]. The barley–Bgh pathosystem represents a paradigm for elucidating crosstalk between cuticular wax and the pathogen and has been instrumental in identifying n-hexacosanal as a genetically defined host recognition signal. In vitro assays by Hansjakob et al. [12] demonstrated that conidia of Blumeria graminis f. sp. hordei (Bgh) exhibit dose-dependent stimulation of conidial germination and appressorium formation on glass surfaces coated with n-Hexacosanal. Moreover, very-long-chain aldehydes ranging from C22 to C30 promoted Bgh pre-penetration development, although efficacy declined as chain length deviated from n-Hexacosanal. By contrast, primary alcohols, alkanes, fatty acids, and esters of identical chain length lacked comparable stimulatory activity [12,18], indicating that the aldehyde functional group is essential for these processes. These studies collectively establish cuticular waxes as key determinants of early powdery mildew development, acting both as physical interfaces and as sources of highly specific chemical cues.

In grapevine, available data indicate both chemical and physical contributions of leaf cuticular waxes to resistance against E. necator. Comparative analysis of cuticular wax compositions between resistant and susceptible grapevine cultivars identified chemical markers associated with resistance, including specific aliphatic alkanes, terpenes, and indole derivatives that directly inhibit conidial germination in E. necator, corroborating the chemical defensive role of leaf cuticular waxes [19]. Physically, a survey of 83 European wild grape accessions revealed a nonlinear threshold relationship between leaf epidermal wax content and powdery mildew resistance, below which susceptibility escalated markedly below a critical wax threshold, yet resistance did not progressively increase beyond this critical point [20]. Microscopic observations further revealed significantly delayed appressorium formation on genotypes exhibiting elevated wax accumulation, which suggests that increased wax loads may modify leaf surface physicochemical properties, including crystal microstructure and surface hydrophobicity, thereby perturbing pathogen development prior to penetration. Collectively, these findings indicate that, unlike the barley–Bgh system where n-Hexacosanal has been defined as a single, major host recognition cue, no comparably specific monomeric signal has yet been established for the grapevine–E. necator interaction. Systematic studies that disentangle the relative contributions of total wax load, crystalline ultrastructure and individual wax monomers to E. necator conidial behavior before penetration remain scarce, particularly in genetically diverse grape germplasm.

To address these gaps, we contrasted four susceptible V. vinifera cultivars with three resistant Chinese wild Vitis accessions and we combined in vivo and in vitro assays with structural and chemical analyses. We established an in vitro germination system for E. necator isolate NAFU1 (En. NAFU1) and then used SEM and GC–MS to compare cuticular wax loads, crystal ultrastructure and chemical composition between susceptible V. vinifera cultivars and resistant Chinese wild Vitis accessions. Finally, we assessed how bulk leaf wax extracts and selected wax monomers influenced conidial germination. Together, these approaches clarified how leaf cuticular waxes shape pre-penetration development of E. necator and provide a mechanistic basis for identifying wax-derived monomers as plant-based leads for eco-friendly, green control of grape powdery mildew.

2. Materials and Methods

2.1. Plant Materials

The grapevines were grown in the grapevine germplasm resources orchard at Northwest A&F University in Yangling, Shaanxi Province, China. Four susceptible V. vinifera cultivars were examined, comprising the table grape ‘Pearl of Csaba’ and the wine grape cultivars ‘Cabernet Sauvignon’, ‘Carignan’, and ‘Pinot Noir’. Three resistant Chinese wild Vitis accessions were also included: V. piasezkii var. pagnucii accession ‘Baishui-40’, V. piasezkii Maxim accession ‘Liuba-6’, and V. romanetii Roman accession ‘Liuba-1’. The disease resistance classifications of these genotypes were adopted from established literature. The susceptible status of ‘Cabernet Sauvignon’, ‘Pinot Noir’, ‘Carignan’, and ‘Pearl of Csaba’ has been documented by Wan et al. [21] and Zhao et al. [22], and the resistant status of ‘Baishui-40’, ‘Liuba-6’, and ‘Liuba-1’ has been documented by Hu et al. [23].

2.2. Extraction of Leaf Cuticular Wax

The leaf cuticular wax was extracted according to the method described by Wang et al. [24] with minor modifications. Mature functional leaves (3rd to 4th node from the base) were sampled from field-grown vines prior to shoot tipping. Leaves were gently rinsed with sterile distilled water, blotted dry with absorbent paper, and leaf discs (3 cm diameter) were punched along the leaf margin using a stainless steel cork borer. Three biological replicates were considered for each sample and each biological replicate consisted of five leaf discs. For wax extraction, five leaf discs were placed in a glass tube containing 20 mL of chloroform and gently agitated for 1 min. The leaf discs were removed with a glass rod, and 30 μL of n-tetracosane (C24) (Macklin Reagent, Shanghai, China) was added as an internal standard. The extract was filtered through qualitative filter paper into a clean 25 mL glass tube, which had been pre-weighed to obtain the tare weight (W₀). The filtrate was concentrated to dryness under a gentle nitrogen stream using a nitrogen evaporator (MD200-2, Allsheng, Hangzhou, China). The tube was then weighed to determine the total wax load gravimetrically. The total area of the five leaf discs (S) was calculated based on the disc diameter (3 cm). Cuticular wax content was calculated as micrograms per square centimeter using the following formula: Wax content (μg/cm²) = (W₁ − W₀)/S, where W₁ is the weight of the tube containing the dried wax residue, W₀ is the tare weight of the empty tube, and S is the total area of the five leaf discs.

2.3. Quantitative and Qualitative Analysis of Leaf Cuticular Waxes by Gas Chromatography—Mass Spectrometry (GC–MS)

The leaf cuticular wax was analyzed according to the method described by Cao et al. [25] with minor modifications. The dried wax residue was redissolved in 1 mL chloroform and transferred into a 2 mL amber GC autosampler vial. After complete drying under nitrogen, 35 μL of pyridine (Macklin Reagent, Shanghai, China) and 35 μL N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (Coolaber, Beijing, China) were added, and the mixture was incubated at 70 °C in a dry block heater for 1 h. The derivatized sample was dried under nitrogen and redissolved in 1 mL chloroform for GC–MS analysis. Wax chemical composition was determined using a gas chromatography–mass spectrometry system (GCMS-2010 SE, Shimadzu, Kyoto, Japan). Splitless injection was employed with the injector temperature maintained at 280 °C. The oven temperature program was as follows: initial hold at 50 °C for 2 min, ramp at 20 °C min⁻¹ to 240 °C with a 2 min hold, ramp at 1.5 °C min⁻¹ to 320 °C with a 15 min hold. The ion source and interface temperatures were set at 250 °C and 280 °C, respectively. Mass spectra were acquired in the range of 50–600 m/z.

2.4. Scanning Electron Microscopy (SEM)

Leaf ultrastructure and epicuticular wax morphology were examined by scanning electron microscopy according to the conventional preparation protocol described by Bensalem-Fnayou et al. [26] for grapevine leaf surfaces, with minor modifications. Mature fully expanded leaves were harvested from the third to fourth node counting from the base of vines grown in the field before shoot tipping. Four susceptible V. vinifera cultivars (‘Cabernet Sauvignon’, ‘Pearl of Csaba’, ‘Pinot Noir’, and ‘Carignan’) and three resistant accessions of Chinese wild Vitis (‘Baishui-40’, ‘Liuba-1’, and ‘Liuba-6’) were examined. Leaf blade segments approximately 5 mm × 5 mm were excised, with major and lateral veins excluded. Tissue samples were initially vacuum infiltrated with 4% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 6.8) at 4 °C for 30 min, then fixed overnight at the same temperature. Subsequently, specimens were rinsed in 0.1 M phosphate buffer for five exchanges of 10 min each, and subjected to a graded ethanol dehydration series comprising 30%, 50%, 70%, and 90% ethanol, with 15 min per step. This was followed by immersion in absolute ethanol for three exchanges of 30 min each, and transfer to isoamyl acetate for two exchanges of 20 min each. After critical point drying with CO₂, specimens were mounted on aluminum stubs with conductive adhesive tape and sputter-coated with gold. Images were acquired using a field emission scanning electron microscope (SU5000, Hitachi, Tokyo, Japan).

2.5. In Vitro Conidial Germination Assay

Conidia of powdery mildew require a hydrophobic substrate for germination; therefore, untreated glass slides do not support conidial germination. To address this limitation, microscope slides were prepared according to the following surface modification protocol. Slides were washed with laboratory detergent, rinsed thoroughly with distilled water, immersed in isopropanol for 10 s, transferred to distilled water for 1 min, and dried in air at ambient temperature. The cleaned slides were then coated uniformly with 40 μL of 1% (w/v) polyvinylpyrrolidone K30 (PVP K-30) (Shanghai Lanji, Shanghai, China) dissolved in chloroform, and allowed to dry in a fume hood for 24 h. Subsequently, 40 μL of 0.5% (w/v) Formvar resin (Electron Microscopy Sciences, Hatfield, PA, USA) (polyvinyl formal dissolved in chloroform) was applied uniformly onto the slides previously coated with PVP K-30, and the slides were dried in a fume hood for 16 h before use.

Conidia were brushed gently from grape leaves exhibiting powdery mildew symptoms and inoculated uniformly onto slides coated with Formvar. Slides were then incubated in darkness at 25 °C and 95% relative humidity for 24 h. Slides prepared without further wax application served as blank controls. The mock (blank control) consisted of coated slides treated with 0.5% Formvar resin solution in chloroform lacking wax monomers or extracts, thereby providing the identical solvent and substrate background while excluding biologically active wax constituents. For wax extract treatments, isolated leaf cuticular wax was dissolved in 0.5% Formvar solution, and the calculated volume was applied to coated slides to match the natural wax concentration on grapevine leaves. For individual wax monomer treatments, components identified by GC–MS were dissolved in 0.5% Formvar resin solution. Working concentrations were calculated from the natural leaf wax content (C, μg/cm²), the effective coating area of the slide (S, 15 cm²), and the coating volume (V, 40 μL). Mass concentration (ρ, mg/mL) was determined as ρ = (C × S)/V, and subsequently converted to molar concentration (C_mol, mol/L) using the molecular weight (M, g/mol) of each component: C_mol = ρ/M. Consistent with the approach of Hansjakob et al. [12], wax monomers were prepared at concentrations of 7 × 10⁻³, 7 × 10⁻⁶, and 7 × 10⁻⁹ mol/L, with each biological replicate containing 150 spores. Standard compounds were obtained from the following suppliers: n-octacosane, n-triacontane, and n-triacontanol were purchased from Aladdin Reagent (Shanghai, China); palmitic acid, stearic acid, n-hexacosane, and n-octacosanol from Macklin Reagent (Shanghai, China); and n-dotriacontanol from Rhawn Reagent (Shanghai, China). Notably, very-long-chain aldehydes (e.g., n-hexacosanal, n-octacosanal, n-triacontanal), although implicated as germination cues in other powdery mildew pathosystems, were excluded from this panel due to the lack of commercially available standards and the specialized organic synthesis required for their preparation.

2.6. Statistical Analysis

Statistical significance was determined using one-way analysis of variance (ANOVA) followed by the LSD multiple comparison test (p < 0.05), whereas identical letters indicate non-significant differences (p > 0.05). Additionally, germination rate data underwent the arcsine transformation. All statistical analyses were performed using SPSS 27.0. Data visualization was conducted using GraphPad Prism 10.1.2, and data organization was performed using Microsoft Excel 2016.

3. Results

3.1. Characterization of In Vitro Germination in En. NAFU1

To establish a robust assay for assessing the influence of cuticular waxes on conidial germination, we first examined the ontogeny preceding host penetration in E. necator isolate NAFU1 on leaf surfaces of V. vinifera cv. Cabernet Sauvignon at 24 h post-inoculation (Figure 1). Trypan blue staining revealed a discrete morphogenetic sequence encompassing six distinct stages: (i) ungerminated conidia (Co); (ii) conidia bearing a short primary germ tube (PGT); (iii) conidia with an elongated PGT; (iv) conidia possessing both primary and secondary germ tubes (SGT); (v) conidia with an appressorial germ tube (AGT); and (vi) conidia with a mature appressorium (Ap). Consistent with established infection biology for powdery mildew, all conidia displaying any of the following: PGT, SGT, AGT, or Ap were classified as germinated. When conidia were deposited onto glass slides coated with Formvar resin and polyvinylpyrrolidone K30 (PVP K-30), the in vitro developmental trajectory recapitulated the in vivo sequence, with congruent morphological characteristics at each corresponding stage. This concordance confirms that the Formvar-based in vitro platform faithfully reproduces natural germination dynamics on the grapevine phylloplane, validating the utility of this system for subsequent wax bioassays.

3.2. Structural and Quantitative Analysis of Leaf Cuticular Wax

To elucidate the structural diversity of leaf cuticular wax among grapevines and its potential impact on conidial germination, we conducted phenotypic (Figure 2A) and ultrastructural (Figure 2B) analyses of four susceptible V. vinifera cultivars (‘Cabernet Sauvignon’, ‘Pearl of Csaba’, ‘Pinot Noir’, and ‘Carignan’) and three resistant Chinese wild Vitis accessions (‘Baishui-40’, ‘Liuba-1’, and ‘Liuba-6’). Scanning electron microscopy (SEM) further revealed pronounced intergroup differences in epicuticular wax crystal morphology, spatial density, and surface topography. Susceptible V. vinifera cultivars exhibited lamellar wax crystals organized into regular arrays with low topographic relief (Figure 2B). In contrast, resistant Chinese wild Vitis accessions ‘Baishui-40’ and ‘Liuba-1’ displayed granular or blocky crystals irregularly aggregated, which generated a spatially complex physical barrier with elevated topographic relief, while ‘Liuba-6’ exhibited an intermediate morphology comprising both lamellar and granular crystals. These results suggest that susceptible V. vinifera cultivars are characterized by smooth lamellar wax structures, whereas resistant Chinese wild Vitis accessions possess dense aggregations of granular and angular crystals, which suggests that interfacial physical properties may influence pathogen colonization.

Gravimetric quantification of total cuticular wax loads revealed that susceptible V. vinifera cultivars accumulated substantially lower wax quantities than resistant Chinese wild Vitis accessions (Supplementary Table S1). Wax loads among the four susceptible V. vinifera cultivars spanned 89.9 to 104.7 μg/cm², whereas values for Chinese wild Vitis accessions were markedly elevated, with ‘Liuba-1’ showing the highest content at 188.3 μg/cm², followed by ‘Liuba-6’ at 141.5 μg/cm² and ‘Baishui-40’ at 127.4 μg/cm² (Figure 3). Consistent with ultrastructural observations (Figure 2B), these quantitative data indicate that susceptible V. vinifera cultivars, despite reduced wax accumulation, are characterized by uniform lamellar crystals with smooth epidermal surfaces. In contrast, ‘Liuba-1’ and ‘Baishui-40’, which exhibited elevated wax loads, displayed dense blocky crystals generating pronounced surface roughness. The accession ‘Liuba-6’, although classified as a Chinese wild genotype with wax contents exceeding those of susceptible V. vinifera cultivars, presented an intermediate crystal morphology, suggesting that wax accumulation and crystalline ultrastructure did not show a simple linear relationship. These findings collectively demonstrate substantial intergenotypic variation in both wax deposition and crystal organization, likely reflecting distinct chemical profiles that potentially govern colonization dynamics of En. NAFU1 conidia on leaf surfaces.

3.3. Influence of Cuticular Wax Extracts on Conidial Germination

To evaluate the direct influence of leaf cuticular wax on conidial germination, extracts from the aforementioned grapevines were applied uniformly to glass slides at native foliar concentrations. The coated substrates were inoculated with En. NAFU1 conidia, and germination rate was evaluated at 24 h post-inoculation (Supplementary Table S2). Relative to the blank control (Mock; 31.6%), wax extracts from all grapevines significantly enhanced conidial germination (Figure 4); although the magnitude of stimulation varied substantially among genotypes, no clear pattern distinguished susceptible V. vinifera cultivars from resistant Chinese wild Vitis accessions. Notably, resistant ‘Liuba-6’ (51.8%) exhibited a stimulatory effect comparable to that of susceptible ‘Pearl of Csaba’ (53.5%), and both susceptible ‘Carignan’ (47.9%) and resistant ‘Liuba-1’ (47.9%) produced equivalent germination rates. Although results show that conidial germination is not attributable to global cuticular wax, this does not discard that global wax may mask the effect of individual components on conidial germination. This possibility prompted us to conduct GC–MS profiling to identify the specific wax constituents responsible for these differential effects.

3.4. Chemical Profiling of Cuticular Wax Constituents

To elucidate the chemical determinants underlying the variable stimulatory activity of wax extracts among the aforementioned grapevines, we profiled leaf cuticular waxes of these genotypes by GC–MS (Supplementary Table S3). Analysis revealed five major compound classes, namely primary alcohols, alkanes, aldehydes, fatty acids, and terpenoids, with each genotype exhibiting a distinct predominant chemical class (Figure 5A). Susceptible V. vinifera cultivars were characterized by elevated primary alcohol contents ranging from 779.1 to 1223.0 μg/dm², whereas resistant Chinese wild Vitis accessions showed higher levels of aldehydes. Although the susceptible cultivar ‘Carignan’ exhibited a relatively high alkane percentage, absolute alkane contents were generally elevated in resistant accessions. Aldehyde contents were substantially higher in resistant Chinese wild Vitis accessions (‘Baishui-40’, 1166.6; ‘Liuba-1’, 3235.4; ‘Liuba-6’, 852.2 μg/dm²) than in susceptible V. vinifera cultivars (134.4 to 253.9 μg/dm²) (Figure 5C). A comparable pattern was evident for alkanes, with the highest content observed in ‘Baishui-40’ (535.6 μg/dm²), followed by ‘Liuba-1’ (202.4 μg/dm²) and ‘Liuba-6’ (96.7 μg/dm²), whereas susceptible V. vinifera cultivars exhibited substantially lower alkane contents (29.7 to 30.1 μg/dm²) (Figure 5E). In contrast, primary alcohol contents in resistant Chinese wild Vitis accessions (‘Baishui-40’, approximately 380 μg/dm²; ‘Liuba-6’, 433.7 μg/dm²) were below those detected in susceptible V. vinifera cultivars (Figure 5B). Furthermore, ‘Liuba-6’ registered the highest terpenoid content (87.8 μg/dm²), which exceeded all other grapevines (Figure 5D), whereas fatty acid contents varied within a narrow range among the aforementioned grapevines, from 130.4 to 234.7 μg/dm² (Figure 5F). These findings reveal systematic divergence in cuticular wax chemical class composition between susceptible V. vinifera cultivars and resistant Chinese wild Vitis accessions, thereby establishing a chemical basis for the differential stimulatory effects on conidial germination observed in vitro.

Subsequent to compound class-level characterization, we further performed quantitative comparisons of individual wax monomers. Primary alcohols comprised five homologues, including n-hexacosanol, n-heptacosanol, n-octacosanol, n-triacontanol, and n-dotriacontanol (Figure 6A), with n-octacosanol, n-triacontanol, and n-dotriacontanol constituting the predominant constituents across all genotypes and n-triacontanol representing the most abundant homologue. ‘Liuba-1’ accumulated significantly elevated levels of n-hexacosanol and n-heptacosanol relative to other genotypes, whereas ‘Liuba-6’ and the former exhibited reduced n-octacosanol and n-triacontanol contents compared with susceptible V. vinifera cultivars. Seven aldehyde homologues, including n-hexacosanal, n-heptacosanal, n-octacosanal, n-nonacosanal, n-triacontanal, n-dotriacontanal, and n-tetratriacontanal, were identified. ‘Baishui-40’, ‘Liuba-1’, and ‘Liuba-6’ all displayed elevated contents across all chain lengths relative to susceptible V. vinifera cultivars, with ‘Liuba-1’ exhibiting particularly pronounced accumulation of n-octacosanal, n-triacontanal, and n-dotriacontanal (Figure 6E). Alkanes included n-hexacosane, n-octacosane, and n-triacontane, except for ‘Carignan’, which registered the highest alkane contents across all chain lengths. Resistant Chinese wild Vitis accessions generally exhibited elevated n-hexacosane, n-octacosane, and n-triacontane levels relative to the remaining susceptible V. vinifera cultivars (Figure 6C). Three terpenoid constituents were identified, including neophytadiene, α-tocopherol, and α-amyrin. Neophytadiene and α-tocopherol exhibited minor intergroup variation. In contrast, ‘Liuba-6’ accumulated α-amyrin at levels that were approximately three to four times higher than those found in susceptible V. vinifera cultivars (Figure 6D). Fatty acids were represented by palmitic acid and stearic acid homologues, with palmitic acid as the dominant constituent (Figure 6B). ‘Liuba-1’ exhibited the highest accumulation of palmitic acid, while stearic acid content was comparable across all genotypes. Primary alcohols (n-octacosanol, n-triacontanol, and n-dotriacontanol), alkanes (n-hexacosane, n-octacosane, and n-triacontane), aldehydes (n-hexacosanal, n-octacosanal, and n-triacontanal), and fatty acids (palmitic acid and stearic acid) were ubiquitously detected in both susceptible V. vinifera cultivars and resistant Chinese wild Vitis accessions, albeit at varying relative abundances, prompting us to evaluate the direct effects of representative shared monomers on conidial germination through in vitro bioassays.

3.5. Effects of Wax Monomer Concentration on Conidial Germination

To evaluate the direct effects of representative wax monomers on conidial germination, we employed primary alcohols (n-octacosanol, n-triacontanol, n-dotriacontanol), alkanes (n-hexacosane, n-octacosane, n-triacontane), and fatty acids (palmitic acid, stearic acid). These monomers were chosen based on three criteria: (i) they were ubiquitously detected across all examined genotypes (Figure 6); (ii) they collectively represent the predominant chemical classes in grapevine leaf cuticular wax (Figure 5); and (iii) authentic standards were commercially available for rigorous concentration-dependent bioassays. Notably, very-long-chain aldehydes (e.g., n-hexacosanal, n-octacosanal, n-triacontanal), although implicated as germination cues in other powdery mildew pathosystems, were excluded from this panel due to the lack of commercially available standards and the specialized organic synthesis required for their preparation. On conidial germination, eight commercially available standards were evaluated at three concentrations (7 × 10⁻³, 7 × 10⁻⁶, and 7 × 10⁻⁹ mol/L) (Supplementary Figure S1). Statistical analysis revealed that conidial germination was significantly affected by both monomer identity and concentration (Supplementary Table S4). Among the alkanes, n-hexacosane exhibited no significant effect at any concentration tested. In contrast, n-triacontane produced a concentration-dependent response featuring suppression at elevated concentrations, with germination rates declining to a minimum of approximately 10% at 7 × 10⁻³ mol/L, followed by progressive recovery to approximately 20% at 7 × 10⁻⁹ mol/L (Figure 7A–C). Within the primary alcohol class, n-octacosanol and n-triacontanol showed no significant effect at any concentration tested, and maintained germination rates between 25% and 35%. In contrast, n-dotriacontanol significantly enhanced germination exclusively at 7 × 10⁻³ mol/L, whereas responses at reduced concentrations comparable to those of the mock control (Figure 7D–F). Among fatty acids, palmitic acid exhibited no significant effect at any concentration. In contrast, stearic acid displayed a characteristic stimulatory response that was concentration-dependent, with germination rates increasing as concentrations declined from 7 × 10⁻³ to 7 × 10⁻⁹ mol/L, achieving levels significantly exceeding those of the mock control at 7 × 10⁻⁶ mol/L (Figure 7G,H). Given that the native foliar concentrations of stearic acid across all genotypes approximated the 7 × 10⁻⁶ mol/L treatment level, these findings indicate that stearic acid likely functions as a principal wax monomer promoting conidial germination of Erysiphe necator in grape leaf cuticular wax.

4. Discussion

Very-long-chain aldehydes residing in plant leaf cuticular waxes serve as potent chemical elicitors of conidial germination in powdery mildews. Among the chemically characterized cuticular wax constituents, aldehydes constitute the chemical determinants essential for conidial prepenetration processes [27,28]. For instance, n-hexacosanal derived from barley leaf surfaces markedly stimulates germination in Blumeria graminis f. sp. hordei [12,29]. Likewise, conidial germination of B. graminis f. sp. tritici exhibits strong dependence on host-derived very long chain aldehydes, predominantly those of n-hexacosanal, n-octacosanal, and n-triacontanal [30]. Genetic evidence from Arabidopsis further substantiates the obligatory role of aldehydes in establishment by obligate biotrophs. The cer3 mutant, defective in very-long-chain aldehyde biosynthesis, presents a leaf surface where prepenetration by Golovinomyces orontii is severely impeded, whereas the cer1 mutant, impaired in alkane synthesis, does not confer this inhibitory effect [14]. Consistent with these findings, the present study detected n-octacosanal, n-triacontanal, and n-dotriacontanal in both susceptible V. vinifera cultivars and resistant Chinese wild Vitis accessions, with resistant accessions exhibiting markedly elevated contents across all chain lengths relative to susceptible cultivars (Figure 6E). Although these aldehydes are well-established germination cues in other powdery mildew pathosystems and their higher abundance in resistant accessions might theoretically enhance conidial germination, the resistance of Chinese wild Vitis accessions is not compromised by this compositional feature, indicating that resistance operates through mechanisms independent of direct wax-mediated inhibition of spore germination.

In addition to aldehydes, this study identified stearic acid as a stimulator of conidial germination, a finding not previously reported for powdery mildew. The biological activity of fatty acids toward fungi is contingent upon pathogen lifestyle, and manifests predominantly as antifungal activity against necrotrophic pathogens. C4–C16 saturated fatty acids as well as linoleic and α-linolenic acids have been shown to inhibit mycelial growth and spore germination in necrotrophic phytopathogens including the early blight fungus (Alternaria solani), the Fusarium wilt fungus (Fusarium oxysporum), and the damping-off fungus (Rhizoctonia solani) [31,32]. Conversely, in the entomopathogenic fungus such as Metarhizium rileyi, oleic and linoleic acids promote conidial germination, appressorium formation, and stress tolerance [33]. Collectively, these findings position stearic acid as a ubiquitous and active stimulator of E. necator conidial germination; The comparable abundance across susceptible and resistant genotypes, coupled with its potent stimulatory effect at physiologically relevant concentrations, suggests that stearic acid may function as a basal chemical signal enabling host recognition, complementary to the established very-long-chain aldehyde pathway. The relative contributions of fatty acids and aldehydes to E. necator host recognition, and whether stearic acid represents a prerequisite cue whereas aldehydes act as enhancing cofactors, remain to be elucidated. We acknowledge that very-long-chain aldehydes, the most established class of powdery mildew germination cues, were not included in our monomer bioassays owing to the lack of commercially available standards and the specialized organic synthesis expertise required for their preparation from corresponding alcohols. Consequently, a direct comparison between stearic acid and very-long-chain aldehydes under identical experimental conditions remains to be performed. Future studies employing authentic aldehyde standards will be necessary to determine the relative contributions of fatty acids and aldehydes to E. necator host recognition and to fully resolve the functional relationship between these distinct chemical signaling pathways.

These results indicate that the resistance of Chinese wild Vitis accessions against E. necator is not attributable to the inhibition of conidial germination at the initial contact stage, but probably to the impediment of post-germination penetration via altered wax crystal architecture and surface roughness, complemented by induced defense responses upon pathogen recognition. This interpretation reconciles the apparent contradiction that cuticular wax extracts from both susceptible V. vinifera cultivars and resistant Chinese wild Vitis accessions uniformly promote conidial germination in vitro, a phenomenon which seems incompatible with the resistance phenotype documented in Chinese wild grapevines. This discrepancy likely reflects distinct resistance mechanisms operating across genotypes. For instance, wax constituents from certain Turkish grape cultivars resistant to powdery mildew, including specific fatty acids, alkanes, and terpenoids, inhibit conidial germination [19], indicating that wax chemistry alone is not the sole determinant of resistance. The resistance mechanisms deployed by resistant Chinese wild Vitis accessions against En. NAFU1 are unlikely to rely on the inhibitory activity of cuticular waxes toward conidial germination; instead, they may be mediated through induced defense responses. In ‘Baishui-40’, resistance is primarily conferred by the REN17 locus, which mediates defense responses through salicylic acid signaling. This includes elevated expression of EDS1, PR1, PAL, PR3, SOD, POD, and CAT, and restricts pathogen invasion and colonization through activation of hypersensitive cell death [22,23]. Beyond the aforementioned hormone signaling and cell defense pathways, some resistant Chinese wild Vitis accessions may alternatively confer resistance through the induced accumulation of secondary metabolites with direct antimicrobial activity. For example, in the Chinese wild species Vitis quinquangularis accession ‘Danfeng-2’, the WRKY transcription factor VqWRKY56 activates salicylic acid signaling to promote localized proanthocyanidin accumulation, generation of reactive oxygen species, and programmed cell death, thereby reinforcing resistance to E. necator [34]. In the present study, ‘Liuba-6’ exhibited specific enrichment of α-amyrin, whereas previous reports have documented high resveratrol content in ‘Liuba-1’. Collectively, these findings suggest that ‘Liuba-1’ and ‘Liuba-6’ deploy antimicrobial defense strategies mediated by secondary metabolites, utilizing resveratrol and α-amyrin as principal effectors, rather than relying on cuticular wax chemistry to suppress conidial germination at initial contact stages.

Terpenoids constitute another major class of wax constituents with biologically variable effects on fungal development. In resistant eucalyptus genotypes, cycloartenol, a triterpenoid enriched in epicuticular wax, along with hexanedioic acid, suppressed conidial germination in Teratosphaeria destructans by more than 75% [35]. Conversely, lupeol, a triterpenoid abundant in the cuticular wax of Ricinus communis fruits, functions as a chemical recognition cue that promotes conidial germination, hyphal growth, and sporulation in Botrytis cinerea [16]. In the present study, α-amyrin, neophytadiene, and α-tocopherol were detected in all genotypes examined, although ‘Liuba-6’ exhibited α-amyrin accumulation at levels approximately threefold to fourfold higher than those in other accessions. Because α-amyrin and lupeol are structurally related triterpenoids with potentially contrasting biological activities, this hypothesis is supported by two lines of evidence: first, α-amyrin is enriched in the epicuticular wax fraction; second, ‘Liuba-6’, which accumulates the highest α-amyrin levels, exhibits an intermediate crystal architecture (Figure 2B). Nevertheless, we acknowledge that this interpretation remains speculative and requires validation through bioassays with purified α-amyrin.

Cuticular resistance is governed by the synergistic interaction among wax chemical composition, cuticle thickness, and epicuticular wax crystal organization, collectively producing the lotus effect [2,9,36,37]. Although the present in vitro assays demonstrate that specific wax monomers, including stearic acid, n-triacontane, and n-dotriacontanol, modulate conidial germination, these chemical properties constitute only one facet of the cuticular defense system. The mechanical barrier against pathogen penetration, which is provided by cuticle thickness, constitutes another critical defensive component. For instance, resistance mediated by the cuticle in grapevine leaves is regulated developmentally, where cuticle thickness in juvenile leaves exhibits a negative correlation with powdery mildew biomass and thereby serves as a principal physical impediment to fungal ingress [38]. Furthermore, the total thickness of the cuticle and wax layers in berries of Chinese wild Vitis accessions resistant to powdery mildew is significantly greater than that in berries of European susceptible varieties, which exhibits a positive correlation with disease resistance [39]. Moreover, surface roughness determined by wax crystal morphology directly modulates appressorium attachment and the efficacy of penetration peg formation. For example, lamellar wax crystals on the adaxial surface of pea leaves facilitate pathogen germination and appressorium differentiation, whereas their removal markedly impairs early pathogen development [40]. In the present study, susceptible V. vinifera cultivars exhibited lamellar crystals, whereas Chinese wild Vitis accessions displayed granular morphologies, with the exception of ‘Liuba-6’, which presented an intermediate architecture comprising both lamellar and granular crystals (Figure 2B). The relative contributions of these divergent crystal organizations, together with cuticle thickness, to resistance against E. necator remain to be determined. A limitation of the present study is the absence of quantitative in vivo data documenting pathogen development on intact leaves of susceptible V. vinifera cultivars and resistant Chinese wild Vitis accessions. Future investigations should employ time-series live inoculation assays to quantify the proportional distribution of each developmental stage preceding host penetration, specifically ungerminated conidia, germ tubes, and appressoria, on leaf surfaces of divergent genotypes. Such data would facilitate a comprehensive assessment of how wax chemical composition, cuticle thickness, and crystal organization collectively modulate the outcome of interactions between grapevine and E. necator.

5. Conclusions

This study demonstrates that the leaf cuticular wax content of susceptible V. vinifera cultivars was lower than that of resistant Chinese wild Vitis accessions. In vitro assays showed that leaf cuticular wax extracts from both susceptible V. vinifera cultivars and resistant Chinese wild Vitis accessions promoted conidial germination of En. NAFU1, These results indicate that resistance in Chinese wild grapes is not mediated by direct wax inhibition of spore germination and instead point to stearic acid as a candidate wax monomer for host recognition by En. NAFU1.