VDAL enhances wheat resistance to Fusarium graminearum by modulating defense signaling and reducing deoxynivalenol accumulation

Abstract

Fusarium head blight (FHB), primarily caused by Fusarium graminearum, is a destructive disease leading to significant yield losses and mycotoxin contamination worldwide. This study investigated the efficacy and molecular mechanism of VDAL (Verticillium dahliae Asp-f2 Like protein), a novel plant immune inducer, in enhancing wheat resistance to F. graminearum. Field trials over two consecutive seasons revealed that three applications of VDAL effectively reduced both disease severity (by > 65%) and deoxynivalenol (DON) accumulation in wheat grains. In vitro assays confirmed that VDAL does not directly inhibit fungal growth. Transcriptomic and qPCR analysis indicated that VDAL’s protection is associated with reduced expression of specific fungal trichothecene biosynthesis genes (TRI9, TRI12) during infection. Strikingly, VDAL orchestrated distinct wheat transcriptional responses conditional on the presence of the pathogen. Under pathogen-free conditions, VDAL enhanced defenses by upregulating genes involved in cuticular wax biosynthesis, flavonoids and terpenoids biosynthesis, and photosynthetic capacity, thereby reinforcing physical and chemical barriers. Upon F. graminearum challenge, VDAL specifically activated a broad-spectrum immune response, including genes associated with pattern-triggered immunity (PTI), salicylic acid (SA)/jasmonic acid (JA)/ethylene (ET) signaling, reactive oxygen species (ROS) generation, calcium flux, cell wall proteins, fungal resistance genes, and systemic acquired resistance (SAR). Importantly, this immune activation remained sustained for at least five days post-application. Key transcription factors integrating hormone signaling (WRKY70, MYB44, TIFY11, ERFs) were identified as central regulators in this process. Our findings indicate that VDAL enhances resistance to FHB by activating sustained host defense responses. The pathogen-dependent induction of defense pathways highlights the precision and efficiency of immune modulation underscores VDAL’s potential as a sustainable biocontrol agent for integrated FHB management.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-026-12964-9.

Highlights

VDAL reduces Fusarium head blight severity and DON accumulation in wheat.

VDAL is associated with reduced expression of specific TRI genes in Fusarium graminearum during infection.

VDAL induces distinct wheat transcriptional responses under pathogen-free and infected conditions.

VDAL enhances SAR via hormone signaling and ROS-related pathways.

VDAL induces sustained immune activation in wheat.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-026-12964-9.

Introduction

Wheat Fusarium head blight (FHB), primarily caused by Fusarium graminearum ranks among the most destructive diseases of wheat and barley worldwide. [1, 2] In addition to causing substantial yield and economic losses, mycotoxins such as deoxynivalenol (DON), produced by F. graminearum on infected grains, pose severe threats to food and feed safety due to its cytotoxicity and inhibitory effects on protein synthesis. [3] Current FHB control heavily relies on chemical fungicides such as tebuconazole, phenamacril, and pydiflumetofen. [4] However, the emergence of resistance in fungal populations [5, 6] coupled with growing public concern over pesticide residues and environmental health, underscore an urgent need to develop sustainable and eco-friendly alternative strategies.

Harnessing the plant’s innate immune system through the application of immune inducers, or elicitors, represents a promising avenue for sustainable crop protection. [7, 8] These agents, which include proteins, oligosaccharides, chitosan, lipids, small molecules, and other compounds, active broad-spectrum resistance without directing antimicrobial activity, thereby minimizing environmental impact and selection pressure for pathogen resistance. [7, 8] Among them, plant immune-activating proteins are of particular interest for their high biological activity and environmental compatibility. [8, 9, 10] For instance, harpin protein based Messenger was commercialized by Eden Biosciences (USA), for disease control in several crops. [10] In China, elicitor proteins like PeaT1 and Hrip1, derived from Alternaria spp., have been developed into bio-pesticides (Atailing) for virus control and growth promotion. [11, 12].

Plants deploy two primary strategies to recognize and resist pathogens: pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Upon pathogen attack, pattern recognition receptors (PRRs) on the plant cell membrane recognize pathogen-associated molecular patterns (PAMPs), triggering PTI, [13] which involves calcium influx, reactive oxygen species generation, activation of MAPK protein kinase cascades, regulation of transcription factors (TFs) such as bZIPs and WRKYs, promotion of callose deposition, resistance gene expression, and changes in hormone levels. [14, 15, 16] PTI serves as the first line of defense against pathogens, halting their further colonization. [17] In response, pathogens secrete effector proteins to undermine PTI, leading to effector-triggered susceptibility (ETS). Simultaneously, plant resistance proteins recognize these effector proteins, triggering ETI, which often results in a hypersensitive response (HR), thus serving as the second line of defense against pathogen invasion. [14].

Plant hormones, such as salicylic acid (SA) and jasmonic acid (JA), play critical roles in plant immune defense. [18] Upon pathogen recognition, pattern recognition receptors (PRRs) activate pattern-triggered immunity (PTI) and induce rapid SA biosynthesis. SA accumulates through isochorismate synthase (ICS) or phenylalanine ammonia-lyase (PAL) pathways, promoting the nuclear translocation of its receptor NPR1. Nuclear NPR1 interacts with TGA TFs to activate pathogenesis-related (PR) genes (PR1, PR2). [19] SA also induces reactive oxygen species (ROS) burst via RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), reinforcing plasma membrane-based defenses. [20] Locally produced SA moves systemically through the vasculature, cooperating with ethylene (ET) signaling to establish systemic acquired resistance (SAR) and conferring broad-spectrum immunity. [21] Conversely, JA signaling requires COI1 receptor-mediated degradation of JAZ repressors, which release MYC2 to induce phytoalexins (e.g., camalexin) and protease inhibitors against necrotrophic pathogens and insect herbivory. [22] While SA predominantly defends against biotrophic pathogens, JA/ET primarily counter necrotrophic pathogens and insects. [18] These hormones display both antagonistic (e.g., SA-mediated suppression of JA signaling) and synergistic effects (e.g., coregulation of defense genes), enabling precise immune modulation through complex crosstalk. [18].

Although hormone-mediated defense mechanisms against FHB in wheat remain incompletely understood, useful insights can be drawn from a number of disparate studies on hormone-dependent defense responses. SA contributes to basal resistance and induces canonical defense-related genes such as the PR family during F. graminearum infection,^23,24 and constitutive expression of the Arabidopsis NPR1 gene in transgenic wheat enhances basal resistance. [25] JA-related genes and biosynthetic pathways are also activated in infected spikes, and repeated methyl-jasmonate (MeJA) treatments can increase resistance, although JA often has stage-dependent effects—promoting susceptibility early but contributing to resistance at later stages. [23, 24] PR1 is widely used as an indicator of SA pathway activation, yet some PR1 paralogs in wheat and other monocots are inducible by exogenous JA or MeJA, demonstrating the variations in hormone responses among species. [26] ET signaling likely further influences host susceptibility, with components such as EIN2 implicated in facilitating colonization. [27] In parallel, cell-wall traits—including pectin modification and arabinoxylan composition—modulate fungal penetration and disease progression. [28] Collectively, these studies show that wheat FHB resistance is governed by a multilayered network integrating hormone signaling and physical barriers.

VDAL (Verticillium dahliae Asp-f2) is a novel plant immune-activating protein derived from V. dahliae. The overexpression of VDAL in Arabidopsis or cotton protects MYB6 from degradation by PUB25 and PUB26, thereby increasing resistance to Verticillium wilt. [29] The VDAL protein formulation is stable, nontoxic, residue free, and soluble. It can trigger both PTI and ETI, activating the innate immunity of plants, inducing resistance, and promoting growth and development, which in turn enhances crop yield and quality. Foliar applications of VDAL has also been reported to promote growth and yield in peanuts and rice, and to enhance resistance against sheath blight in rice and Sclerotinia sclerotiorum in Chinese cabbage. [29–32] In Chinese cabbage, VDAL was shown to reprogram hormone signaling pathways during the early stages of S. sclerotiorum infection. [32] However, its potential efficacy against the agronomically crucial FHB in wheat, its potential impact on the mycotoxin DON, and any molecular mechanisms underlying its action, particularly its long-term effects, remain entirely unexplored.

In this study, we observed the protective effect of VDAL against FHB in wheat under field conditions and employed multi-transcriptome sequencing to decipher the molecular mechanisms at play. Our results demonstrate that VDAL enhances wheat defense against FHB and DON contamination by activating the plant’s immune system in a pathogen-dependent manner: enhancing basal defense under non-infected conditions and unleashing a full-scale immune attack upon pathogen challenge, while simultaneously suppressing fungal TRI gene expression. This work establishes VDAL as a highly promising candidate for the sustainable management of FHB.

Materials and methods

Materials and pathogen cultures

0.4% VDAL soluble power (SP) was sourced from Pherobio Technology Co.Ltd. (China), while 25% phenamacril suspension concentrate (SC) and 48% phenamacril-tebuconazole SC were obtained from Jiangsu Pesticide Research Institute Co., Ltd. (China). The 40% prothioconazole-tebuconazole SC was supplied by Liyang Zhongnan Chemical industry Co., Ltd. (China). 43% Tebuconazole SC was provided by Yancheng Limin Agrochemical Co., Ltd., China. The wheat cultivars used in this study were Huaimai 20 (2022), developed by the Huai’an Academy of Agricultural Sciences in Jiangsu Province, and Yangmai 15 (2023), and developed by the Lixiahe Regional Institute of Agricultural Sciences in Jiangsu Province.

The F. graminearum wild-type strain PH-1 (NRRL 31084) stored in Institute of Plant Pathology, Institute of Jiangsu Academy of Agricultural Sciences, maintained on PDA plates with a 12-h-light and 12-h-dark cycle at 25 °C. Conidia were produced in the carboxymethyl cellulose (CMC) liquid medium (15 g carboxymethyl cellulose, 1 g yeast extract, 0.5 g MgSO₄, 1 g NH₄NO₃, 1 g KH₂PO₄ and 1 L water) shaken at 175 rpm, 25 °C for 5 days and then collected from 5-day-old CMC medium by filtration and centrifugation. The collected conidia cultured in liquid YEPD (1% yeast extract, 2% peptone, 2% glucose and 1 L water) medium shaken at 175 rpm, 25 °C was used for germination and then for mycelia collection. [33] 0.4% VDAL SP was added to PDA plates at final concentrations of 1 µg/mL, 10 µg/mL and 50 µg/mL for colony morphology observation.

Field plot experiments

Field trials were conducted in 2022 and 2023 at the experimental farm of the Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu Province, China. The experimental field was established as a FHB disease nursery, in which uniform and sufficient disease pressure was ensured by spray inoculation with a conidial suspension of F. graminearum (5 × 10⁴ spores/mL) at the wheat flowering stage. All experiments followed a single-factor randomized complete block design, with three biological replicates per treatment (i.e., three independent plots). Each plot covered an area of 2.5 m².

In 2022, the wheat cultivar ‘Huaimai 20’ was used, and three treatments were established: (1) water-treated control (no chemical application); (2) a VDAL treatment, in which 0.4% VDAL SP was applied by foliar spraying at the booting stage (Zadoks GS 45) and again at early flowering (Zadoks GS 61), with a formulation rate of 150 g/ha for each application; and (3) a chemical control, in which 25% phenamacril SC was applied once at early flowering (Zadoks GS 61) at a rate of 1500 mL/ha.

In 2023, the wheat cultivar ‘Yangmai 15’ was used, and four treatments were included: (1) water-treated control; (2) a chemical control, consisting of a single foliar application of 48% cyanoenoxazole–tebuconazole SC at early flowering (Zadoks GS 61) at a rate of 750 mL/ha; (3) a VDAL treatment, in which 0.4% VDAL SP was applied at the booting stage (GS 45), heading stage (GS 55), and early flowering (Zadoks GS 61), at 150 g/ha for each application; and (4) a combined VDAL and fungicide treatment, in which 0.4% VDAL SP (150 g/ha) was applied at the booting (Zadoks GS 45) and heading (Zadoks GS 55) stages, followed by a mixed application of 0.4% VDAL SP (150 g/ha) and 43% tebuconazole SC (90 mL/ha) at early flowering (Zadoks GS 61). For all treatments, including the water control, the spray volume was standardized at 225 L/ha.

Approximately 5 h after the chemical application at the early flowering stage (Zadoks GS 61), all plots were uniformly sprayed with a mixed conidial suspension of F. graminearum and F. asiaticum at a concentration of 5 × 10⁴ spores/mL, using a spray volume of 225 L/ha. These two species represent the predominant causal agents of FHB in the Nanjing region.

Field disease survey and statistical analysis

At the late milk stage (Zadoks GS 77) of wheat in the field trials described in Sect. Field plot experiments, sampling was conducted at five points per plot, 20 spikes were investigated per point, and the diseased area of the spikes was graded on the basis of the percentage of the spike area affected by the disease; the number of diseased spikes in each grade and the total number of surveyed spikes were recorded. Disease severity indices are presented as mean ± standard deviation (SD). Data were analyzed using analysis of variance (ANOVA) based on a randomized complete block design, with treatment as a fixed effect and block as a random effect; when treatment effects were significant, multiple comparisons were performed using Duncan’s new multiple range test. All statistical analyses were conducted using SPSS version 26.0 software, with the significance level set at P < 0.05.

The grading criterion was as follows: Grade 0: no disease on the entire spike. Grade 1: Diseased spike area is less than 1/4 of the entire spike area. Grade 3: Diseased spike area is 1/4 to 1/2 of the entire spike area. Grade 5: Diseased spike area is 1/2 to 3/4 of the entire spike area. Grade 7: Diseased spike area is more than 3/4 of the entire spike area.

The disease index (DI) is calculated by multiplying the sum of the products of the number of spikes in each grade and their corresponding grade values by 100, divided by the total number of spikes surveyed multiplied by 7.

Control efficiency was calculated by taking the difference between the disease index of the control plot and the disease index of the treated plot, divided by the disease index of the control plot, and then multiplied by 100 to express the result as a percentage.

RNA-seq sample collection

Samples for wheat transcriptome sequencing were collected from the 2023 field experiment, and the wheat cultivar ‘Yangmai 15’ was used. At early anthesis (Zadoks GS 61), after completion of chemical treatments and prior to field spray inoculation, randomly selected spikes were subjected to artificial single-floret point inoculation.

Four treatment groups were established for RNA-seq analysis: (1) CK, water-treated control without pathogen inoculation, sampled from field treatment 1; (2) VDAL, spikes treated with 0.4% VDAL SP without pathogen inoculation, sampled from field treatment 3; (3) Inf, spikes inoculated with F. graminearum without VDAL treatment, obtained by artificial inoculation of spikes from field treatment 1; and (4) InfVD, spikes treated with VDAL and subsequently inoculated with F. graminearum, obtained by artificial inoculation of spikes from field treatment 3.

For the Inf and InfVD groups, single-floret point inoculation was performed using a micropipette to inject a conidial suspension of F. graminearum (2.0 × 10⁵ spores/mL) into eight central spikelets per spike, with 10 µL applied per spikelet. Inoculating eight spikelets per spike was also carried out to ensure adequate fungal biomass.Ten spikes were inoculated per plot. After inoculation, spikes were covered with plastic bags for 48 h to maintain high humidity. Samples were collected at 5 days post inoculation (dpi) by excising the middle portion of each spike (including the inoculated region) with sterile scissors. For the CK and VDAL groups, non-inoculated spikes were sampled at the corresponding positions.

For each treatment group, tissue from ten spikes collected within the same plot was pooled to constitute one biological replicate. Three independent biological replicates were included per treatment, corresponding to three separate field plots.

Fungal transcriptome analysis was conducted on samples from infected wheat tissues (Inf and InfVD), as well as on fungal mycelium cultured in YEPD medium with or without VDAL treatment (MycVD and Myc).

For the Myc and MycVD groups, conidia were inoculated into liquid YEPD medium and incubated at 25 °C with shaking at 175 rpm for 12 h to allow germination. VDAL was then added to the YEPD cultures for the MycVD group at a final concentration of 1 µg/mL, and incubation continued for an additional 4 h. For the Myc group, cultures were incubated under the same conditions for a total of 16 h without VDAL addition. Mycelia were harvested by filtration. Each fungal samples was derived from an independent shaking flask and considered one biological replicate, with three independent replicates per treatment.

To enable simultaneous analysis of host–pathogen transcriptional regulation during infection, plant samples from the Inf and InfVD groups were subjected to RNA-seq analysis for both wheat and F. graminearum. All RNA-seq experiments included three independent biological replicates.

RNA extraction, library construction, sequencing and bioinformatics analysis

Total RNA from both plant and fungal samples was extracted using TRIzol^® reagent (Invitrogen, USA). All RNA samples were treated with DNase I. RNA concentration was measured using the Qubit^® RNA Assay Kit (Life Technologies). RNA integrity and purity were assessed using the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) and NanoPhotometer^® spectrophotometer (IMPLEN, CA, USA), respectively. A total amount of 3 µg RNA per sample was used as input material for the RNA sample preparation. The RNA-seq libraries were prepared with the NEBNext^® Ultra™ RNA Library Prep Kit for Illumina^® (NEB, USA) following the manufacturer’s recommendations and sequenced with an Illumina Nova seq6000 with a paired-end 2 × 150 bp model at Genepioneer Biotechnologies (Nanjing, China). For each replicate, at least 24 Mb paired-end reads were obtained. The resulting RNA-seq reads were mapped onto the reference genomes of Chinese Spring Triticum aestivum and F. graminearum strains PH-1 [34, 35] via HISAT2. [36] The number of reads (count) mapped to each gene/transcript was calculated via Salmon. [37] The Inf3 sample was excluded from the analysis due to its poor reproducibility compared with other samples (Fig. S3B). Differential expression analysis of genes was performed via the DESeq2 package [38] with the exactTest function. Genes with an adjusted p value of less than 0.05 and a |log2FC (log2-fold change) | of greater than 1 were regarded as differentially expressed genes. The GO enrichment results were analyzed via BLAST2GO (www.blast2go.com). KEGG enrichment analysis was performed via TBtools. [39] The datasets generated during the present study are available from NCBI under BioProject accession number PRJNA1181034.

Quantitative real-time PCR (qRT‒PCR)

For the qPCR analysis, total RNA was extracted from samples of the CK group (plants only, no inoculation or VDAL treatment), the InfVD group (plants and fungi, inoculated with F. graminearum after treatment with 0.4% VDAL SP), and the Myc group (fungi only, F. graminearum). The sample collection and processing methods were consistent with those described for the RNA-seq samples (Sect. RNA-seq sample collection). RNA extraction followed the protocol detailed in Sect. RNA Extraction, Library Construction, Sequencing and Bioinformatics Analysis. cDNA was synthesized using the Hifair^® III 1st Strand cDNA Synthesis SuperMix (Yeasen, China). The wheat actin and F. graminearum β-tubulin genes were used as internal controls for the plant and fungal samples, respectively, [40] and the relative expression of each target gene was calculated using the 2^−△△Ct method. For plant samples, each biological replicate consisted of pooled tissue from five independently inoculated spikelets (n = 3). For fungal mycelial samples, each biological replicate was derived from an independent culture (n = 3). All qRT-PCR reactions were performed in technical triplicates. The means and standard deviations of the expression levels were calculated based on the biological replicates. All genes and their corresponding primer sequences used for qPCR in this study are detailed in Supplementary Table 7.

DON extraction

The grain samples for DON quantification were obtained from the field trial described in Sect. Field plot experiments. Twenty spikes of mature wheat were randomly collected from each plot, and the collected spikes were threshed via a small thresholding machine. The grains are then ground into flour via a FOSS cyclone sample mill (CT293). Subsequently, 2.5 g of flour was weighed into a 50 mL centrifuge tube, and an acetonitrile-water mixture (acetonitrile: water = 84:16) was added at a volume four times greater than that of the sample. The mixture was soaked for 4 h and shaken every 30 min. The mixture was centrifuged at 3500 r·min^− 1 for 6 min, and then, 4 mL of the supernatant was collected and passed through a Bond Elut Mycotoxin (Agilent) purification column, allowing it to filter naturally. Two milliliters of the filtrate was transferred into a 15 mL centrifuge tube and dried under a stream of nitrogen gas. Then, 15 mL of a methanol: water mixture (methanol: water = 20:80) was added. The samples were filtered through a 0.2 μm organic microporous membrane, collected in a sample vial, and subjected to high-performance liquid chromatography analysis.

DON was detected using a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) system (Agilent 1290 Infinity LC system coupled to a Triple Quad 6500 Plus, Sciex, USA). The analytical column was an Agilent ZORBAX SB-C18 column (2.1 × 150 mm, 3.5 μm) (Agilent, USA), and the column temperature was maintained at 40 ℃. The flow rate was 0.3 mL/min, and the injection volume was 5 µL. The mass spectrometric analyses were performed with the following operating parameters: gas temperature, 450 ℃; gas flow rate, 10 L/min; nebulizer gas pressure, 55 psi; and capillary voltage, -4500 V. Nitrogen was used as the nebulizing and collision gas. Mycotoxins were analyzed via multiple reaction monitoring (MRM).

Results

VDAL treatment reduces Fusarium head blight (FHB) severity and DON accumulation

After three days of cultivation on PDA plates, no significant differences were observed in the colony growth rate of the F. graminearum PH-1 strain under VDAL-treated versus untreated conditions (Fig. S1), indicating that VDAL does not directly affect the vegetative growth of the pathogen.

Field plot experiments were conducted over consecutive growing seasons (2022–2023) to evaluate the efficacy of VDAL against FHB. In 2022, VDAL was applied twice, during the booting and flowering stages, and was assessed 25 dpi. Although VDAL application did not significantly protect against FHB compared with the control, it resulted in a notable reduction in deoxynivalenol (DON) production (Table 1). In 2023, an additional application was included, with VDAL being applied at the booting, heading, and flowering stages. The three-time VDAL application significantly suppressed both FHB incidence and DON contamination at 25 dpi, resulting in a > 65% reduction in the disease index relative to that of the controls. Furthermore, we included an additional treatment where VDAL was applied three times, with the final application combined with a low dose of 43% tebuconazole SC (90 mL/ha). The efficacy of this combination treatment showed no significant difference compared to three applications of VDAL alone. The chemical control, 48% phenamacril-tebuconazole SC, achieved a control efficacy of 87.43%, which was significantly higher than that of all other treatments. (Table 2). These results further support the ability of VDAL to effectively mitigate FHB and mycotoxin production, particularly when it is applied three times.

Table 1.

The effectiveness of VDAL in the control of Fusarium head blight in 2022

Treatments	Application Date	DON (ug/g)	Disease Index	Control Efficiency (%)
1	--	1.60 ± 0.60a	7.10 ± 3.38a	--
2	0.4% VDAL SP, Booting stage & flowering stage (150 g/ha)	0.95 ± 0.31b	6.24 ± 2.20a	12.10
3	25% Phenamacril SC, flowering stage (375 mL/ha)	1.57 ± 1.04a	1.52 ± 0.30b	78.52

Different letters after the same column of data in the table indicate significant differences at the P < 0.05 level as tested by the least significant difference (LSD) method

Table 2.

The effectiveness of VDAL in the control of Fusarium head blight in 2023

Treatments	Application Data	Disease Index	Control Efficiency (%)	DON (ug/g)
1, Control	--	7.00 ± 1.97a	--	3.60 ± 0.48a
2, 48% Phenamacril·tebuconazole SC	flowering stage (270 mL·90 mL/ha)	0.88 ± 0.68b	87.43	0.62 ± 0.56c
3, 0.4% VDAL SP	booting stage & full heading stage & flowering stage (150 g/ha),	2.31 ± 1.84b	67.00	2.10 ± 0.72b
4, 0.4% VDAL SP, and 43% tebuconazol SC	booting stage & full heading stage (150 g/ha 0.4% VDAL SP), flowering stage (150 g/ha 0.4% VDAL SP and 90 mL/ha 43% tebuconazol SC)	2.14 ± 1.76b	69.43	1.95 ± 0.15b

Different letters after the same column of data in the table indicate significant differences at the P < 0.05 level as tested by the least significant difference (LSD) method

Wheat transcriptome sequencing and differential expression analysis

To elucidate the mechanism underlying VDAL functions in wheat resistance to FHB, spike samples were collected at 5 dpi for wheat transcriptomic analysis. These samples included water control (CK), F. graminearum inoculation (Inf), VDAL treatment (VD), and combined VDAL and F. graminearum inoculation (InfVD). Following the filtration of low-quality reads and adapter sequences, a total of 686,181,348 clean reads, amounting to 191 Gb of clean bases, were obtained (Table S1). The minimum correlation among the three replicates was 84% (Fig. S2). Principal component analysis (PCA) of the 12 arrays revealed dynamic transcriptomic changes in wheat in response to both F. graminearum and VDAL treatment (Fig. S3). Compared with the control, F. graminearum inoculation resulted in the upregulation of 111,31 genes and the downregulation of 2,901 genes. In contrast, VDAL treatment led to the upregulation of 5,195 genes and the downregulation of 3,017 genes (adjusted p value ≥ 5, |log2FoldChange| ≥ 1). Concurrent VDAL and pathogen treatment resulted in the upregulation of 14,684 genes and the downregulation of 7365 genes. A detailed list of differentially expressed genes (DEGs) is presented in Fig. 1 and Table S2.

Fig. 1.

A Volcanic plot of the Inf DEGs relative to the CK DEGs. B Volcanic plot of VDAL DEGs relative to those in the CK. C Volcanic plot of InfVD DEGs relative to those in CK. Inf, VDAL, InfVD samples from infection-stage wheat heads, wheat heads treated with VDAL, and infection-stage wheat heads treated with VDAL, respectively

KEGG enrichment analyses of DEGs induced by F. graminearum or VDAL

KEGG analysis of CK and Inf samples revealed that pathogen infection upregulated genes involved in plant metabolism and the immune response, triggering physiological and defense responses. Twenty KEGG pathways were significantly enriched among the upregulated genes, with the top seven pathways being phenylalanine, tyrosine, and tryptophan biosynthesis (00400); phenylalanine metabolism (00360); glutathione metabolism (00480); tyrosine metabolism (00350); alpha‒linolenic acid metabolism (00592); the MAPK signaling pathway (04016); and ubiquinone and other terpenoid‒quinone biosynthesis (00130), each exhibiting an enrichment factor exceeding 2.5 (Fig. 2A). Among the downregulated genes, the most enriched pathways (q value = 0, enrichment factor > 4) included ATP-dependent chromatin remodeling (03082), the prokaryotic defense system (02048), motor protein (04814), chromosome (09126), chromosome and associated proteins (03036), DNA replication proteins (03032), and starch and sucrose metabolism (00500) (Fig. 2B). In summary, the enriched pathways were associated primarily with metabolism, plant‒pathogen interactions, and the MAPK signaling pathway, all of which are directly linked to pathogen infection. Moreover, metabolic pathways involved in phenylpropanoid biosynthesis, phenylalanine biosynthesis, and glutathione metabolism play crucial roles in antioxidant defense and disease resistance. In contrast, the downregulated pathways included mainly chromatin remodeling and DNA replication. These findings suggest that during pathogen infection, pathways related to plant metabolism, immunity, and disease resistance are activated, whereas pathways associated with chromatin maintenance and DNA repair are suppressed.

Fig. 2.

A KEGG classifications of upregulated DEGs in the Inf group relative to those in the CK group. B KEGG classifications of downregulated DEGs in the Inf group relative to those in the CK group. C KEGG classifications of upregulated DEGs in VDAL relative to those in CK. D KEGG classifications of downregulated DEGs in VDAL relative to those in CK. The vertical axis represents the pathway name, and the horizontal axis represents the enrichment factor

To elucidate the functional gene sets induced by VDAL in wheat, we conducted KEGG enrichment analysis of DEGs under VDAL-only treatment compared with those under CK. The top 20 enriched pathways among the upregulated genes included those related to carbohydrate and protein metabolism, such as starch and sucrose metabolism, galactose metabolism, photosynthesis-antenna protein biosynthesis, and protein metabolism. Additionally, pathways related to stress response, including cutin, suberin and wax biosynthesis; plant hormone signal transduction; phenylpropanoid biosynthesis; flavonoid biosynthesis; ubiquinone and other terpenoid-quinone biosynthesis; cytochrome P450; and tropane, piperidine and pyridine alkaloid biosynthesis, were also significantly enriched (Fig. 2C, D). Interestingly, pathways directly linked to pathogen infection (plant‒pathogen interaction and MAPK signaling) were not enriched in the VDAL-only treatment. These results indicate that VDAL application likely enhances plant physicochemical defenses by activating the cutin/suberin/wax biosynthesis and phenylpropanoid, flavonoid, terpenoid, and alkaloid pathways while concurrently promoting grain nutrient accumulation through the upregulation of carbohydrate metabolism- and photosynthetic antenna protein-related genes.

VDAL specifically actives immune-related pathways under pathogen infection conditions

To further elucidate the regulatory mechanisms activated by VDAL during pathogen infection, we conducted an overlapping analysis of the CK-VS-Inf, CK-VS-VDAL, and CK-VS-InfVD datasets. A Venn diagram analysis revealed both unique and shared DEGs among the three groups (Fig. 3A). A total of 2,461 DEGs were identified as common across all three groups. The Inf-VS-CK comparison revealed 1,585 specific DEGs, comprising 822 upregulated and 763 downregulated genes, but no significant enrichment of specific pathways was detected (Fig. 3A and B, Table S3). In the VDAL-VS-CK comparison, we identified 4,273 specific DEGs, with 2,886 upregulated genes involved in gibberellin and abscisic acid biosynthesis, jasmonic acid signaling, and starch biosynthesis (Fig. 3A and B, Table S3, Table S4). Conversely, 1387 downregulated DEGs were linked to processes such as salicylic acid (SA), chitin, hydrogen peroxide, fungus, and brassinosteroid-mediated signaling pathways (Table S4).

Fig. 3.

A Venn diagram of DEGs in Inf-VS-CK, VDAL-VS-CK, and InfVD-VS-CK. B Volcanic plot of Inf, InfVD, and VDAL relative to CK. The X-axis depicts the fold change in gene expression, where the smaller the value is, the greater the expression relative to CK. The y-axis denotes the false discovery rate, where the larger the value is, the smaller the ratio of the number of false positives to the total number of positives. Red and blue dots represent significantly upregulated and downregulated DEGs, respectively

In the InfVD-VS-CK group, 8,550 specific DEGs were identified, comprising 3860 upregulated and 4,690 downregulated genes (Fig. 3A and B; Table S3). Among the upregulated genes, 977 participated in the chitin response, salicylic acid signaling, MAP kinase phosphatase activity, the fungal response, and jasmonic acid signaling, whereas more than 200 were related to the high light intensity response, ethylene signaling, and heat acclimation (Fig. 4A, Table S5). Notably, the downregulated genes were enriched primarily in hypersensitive response, auxin homeostasis, mismatch repair, and chromatin remodeling (Fig. 4B). These enrichment profiles suggest that VDAL primarily activates plant immunity signaling against pathogens through plant hormone signaling (SA/JA), MAPK, and fungal response pathways.

Fig. 4.

KEGG enrichment analysis of DEGs in the InfVD-VS-CK comparison. A Upregulated genes. B Downregulated genes. The size of the dots represents the enrichment factor range, and the color of the dots represents the number of enriched genes. The enrichment factor represents the ratio of the number of DEGs annotated to the number of KEGG pathways to the total number of DEGs

Gene expression analysis of defense-related genes

To further investigate the genes specifically induced during cotreatment with VDAL and F. graminearum, we performed a heatmap analysis of the upregulated DEGs. This analysis revealed the induction of 21 TFs, including TIFY (TIFY11), ERF (ERF4, ERF54, and RAP2-3), WRKY (WRKY2, WRKY19, WRKY70, and WRKY76), and MYB44, along with three zinc finger protein-encoding genes (LOC123126000, LOC123108228, and LOC123117492), notably ZAT12 (Fig. 5A). The upregulated genes also included pathogenesis-related 1 (PR1), cytochrome p450 72A15-like, E3 ligases ATL6 and RDUF1, peroxisomal acyl-coenzyme An oxidase 1-like ACX1, alpha-dioxygenase DOX1, BAG family molecular chaperone regulator BAG6, SA-binding protein SABP2, gibberellin receptor GID1, U-box domain-containing protein PUB26, UDP-glucosyltransferase UGT13248, serine/arginine-rich splicing factor SR45, MFS transporter ZIFL1, and probable polyamine oxidase PAO2 (Fig. 5A). These genes play critical roles in SA- and JA-mediated signaling pathways, as well as in regulating innate immunity and disease resistance. Furthermore, the heat shock proteins Hsp20, Hsp70, Hsp90, the chaperone protein ClpB1, proteins involved in the early response to dehydration ERD7, the heat stress-associated protein HSA32, and the stress-upregulated protein Nod19 were also significantly induced (Fig. 5B). Collectively, these findings suggest that upon fungal infection, VDAL specifically induces the expression of hormone-related molecules, reactive oxygen, stress response genes, and immune-related proteins, thereby activating plant defense signaling and enhancing resistance to F. graminearum.

Fig. 5.

Relative expression of differentially expressed genes among CK, VD, and InfVD samples. A Immunity-related genes. B Stress-related genes. The colors blue, yellow and red indicate low, medium, and high expression patterns of genes, respectively. CK indicates mock-treated plants, VD indicates VDAL-treated plants, and InfVD indicates plants treated with VDAL and infected with F. graminearum

In addition, we identified 43 upregulated genes associated with calcium signaling (20) and cell wall reinforcement (23), including those encoding the following: (i) cell wall protein GP1; (ii) calcium-dependent protein kinases (CDPK9/16); (iii) calcineurin-like phosphoserines; (iv) calcium-binding CML proteins (CML15/16/21/25/35/49); (v) calcium transporters (uniporter protein 6, sodium/calcium exchanger NCL2, cation/calcium exchanger 1, calcium uptake regulator); and (vi) protein phosphatase 2 C isoforms. These upregulated genes (detailed in Table S6) collectively indicate enhanced calcium-mediated signaling and cell wall fortification pathways.

Quantitative reverse transcription PCR (qRT‒PCR) validation

To validate our RNA-seq findings, qRT‒PCR was performed on genes involved in jasmonic acid signaling, the high light intensity response, the chitin response, and aleurone grain membrane components (Fig. 6). Consistent with the RNA-seq results, VDAL treatment during infection induced the upregulation of these genes, further supporting the hypothesis that VDAL enhances wheat immune responses and disease resistance by activating the jasmonic acid signaling and chitin response pathways. Additionally, VDAL treatment enhances the plant response to high light intensity and promotes the expression of genes related to the aleurone grain membrane, thereby facilitating the accumulation of photosynthetic products and protein storage in wheat grains, leading to improved quality and yield.

Fig. 6.

Gene expression of InfVD-VS-CK. A Genes involved in jasmonic acid-mediated signaling pathways. B Genes involved in the regulation of the highlight intensity response. C Chitin response-related genes. D Related aleurone grain membrane components. CK indicates blank wheat heads, and InfVD indicates infection-stage wheat heads

Transcriptional regulation of F. graminearum by VDAL

Fungal transcriptome analysis was conducted on samples from inoculated wheat tissues (Inf, InfVD), and the others (Myc and MycVD) from F. graminearum grown on YEPD. A total of 181.12 Gb of sequence data were generated, consisting of 458,071,898 Mb reads across 12 samples, with an average of 38,172,658 Mb reads per sample (Table S1, Fig. S3A).

Venn diagram analysis revealed 539 DEGs in F. graminearum during mycelial growth under VDAL treatment versus untreated controls. Among these genes, 210 were VDAL specific, with 107 upregulated and 103 downregulated (Fig. 7A and B; Table S4). Notably, most of these genes were not functionally annotated or associated with core pathogenicity, indicating a minimal impact on essential fungal processes. Interestingly, among the upregulated genes, FG_01G21721, FG_01G05619, FG_01G13739, and FG_01G14741 exhibited considerable changes in expression, with fold changes from 2⁵ to 2¹⁴ (Fig. 7D). Structural domain predictions suggest that FG_01G21721 and FG_01G05619 encode signal peptide proteins, that FG_01G13739 is a glutathione transferase, and that FG_01G14741 contains a putative FMN-binding domain (Fig. 7C). Overall, while VDAL appears to exert minimal influence on the essential life processes of F. graminearum, it does demonstrate some level of recognition and interaction with F. graminearum, an immune protein derived from V. dahlia.

Fig. 7.

Analysis of the RNAseq data of F. graminearum. A Venn diagram of DEGs associated with Inf-VS-Myc, MycVD-VS-Myc, and InfVD-VS-Myc. B Volcanic plot of Inf, InfVD, and MycVD relative to Myc. The X-axis depicts the fold change in gene expression, where the smaller the value is, the higher the expression relative to Myc. The y-axis denotes the false discovery rate, where the larger the value is, the smaller the ratio of the number of false positives to the total number of positives. Red and blue dots represent significantly upregulated and downregulated DEGs, respectively. C Schematic diagram of the protein sequence. D Abundances of the four-gene transcripts in Myc and MycVD based on RNA-seq data. FPKM: Fragments per kilobase of exon per million fragments mapped. Inf, InfVD, and MycVD samples from infected-stage wheat heads, infection-stage wheat heads treated with VDAL, and vegetative mycelia treated with VDAL, respectively

VDAL induces the downregulation of two DON biosynthesis genes in F. graminearum during infection

Field trial results indicate that VDAL can partially reduce the incidence of Fusarium head blight and reduce the production of DON during spike infection. However, the precise mechanism behind this reduction in DON production remains unclear. RNA-seq analysis revealed that TRI genes were significantly upregulated during the infection stage in wheat compared with those in the control mycelium. Notably, VDAL application during this stage led to a partial downregulation of TRI gene expression relative to that in the Inf sample, although the expression levels remained higher than those observed during the mycelial stage (Fig. 8A). qRT‒PCR analysis of the TRI genes TRI1, TRI101, TRI9, TRI12, and TRI14 demonstrated that all these genes were significantly induced during infection. However, compared with those in the Inf sample, the expression levels of TRI1, TRI101, and TRI14 were not significantly different, whereas the expression levels of TRI9 and TRI12 were significantly lower in the InfVD sample (Fig. 8B). These results suggest that VDAL treatment during infection is associated with reduced expression of TRI9 and TRI12. This downregulation may partially contribute to the reduced DON accumulation, although the precise mechanism requires further investigation.

Fig. 8.

The expression levels of the TRI genes in Myc, Inf, and InfVD. A Abundances of TRI gene transcripts in Myc, Inf, and InfVD based on RNA-seq data. FPKM: Fragments per kilobase of exon per million fragments mapped. Analysis of variance (ANOVA) was performed for significant differences in the expression levels of the TRI genes (based on FPKM values from three biological replicates) between the Inf and InfVD groups, *p < 0.05, **p < 0.01. B Expression levels of TRI1, TRI101, TRI9, TRI12, and TRI14 in Myc, Inf, and InfVD. Relative expression was calculated using the 2^{−ΔΔC t} method. The relative expression level of each gene in Myc was set to 1. The means and standard errors were calculated from data from three independent biological replicates. ANOVA was performed using SPSS to determine whether there were statistically significant differences in expression levels among the different treatment groups, *p < 0.05, **p < 0.01. Myc, Inf, and InfVD indicate vegetative mycelia, F. graminearum infection, and F. graminearum infection and VDAL treatment, respectively

Discussion

VDAL does not directly affect F. graminearumgrowth but significantly mitigates FHB and mycotoxin accumulation in wheat

Our initial in vitro assays confirmed that VDAL did not inhibit the mycelial growth of F. graminearum (Fig. S1), aligning with its definition as a plant immune inducer rather than a direct antifungal agent. Transcriptomic analysis of VDAL-treated F. graminearum mycelia further supported this, showing no significant disruption of core developmental or pathogenic pathways. The induction of a few genes encoding putative secreted proteins (Fig. 7C) suggests a potential interaction between VDAL and the fungus, possibly triggering subtle responses that do not compromise viability, a notion that warrants further investigation.

Previous studies have demonstrated that the exogenous application of VDAL can reduce the incidence of sheath blight and improve yield in rice. [31] In B. rapa, VDAL regulates the ABA, auxin, and ethylene signaling pathways to increase innate immunity, thereby reducing the occurrence of sclerotinia rot. [32] Our field-trail investigation confirmed that three repeated applications of VDAL effectively reduce FHB severity and mycotoxin levels.

VDAL does not affect the mycelial growth of F. graminearum but significantly reduces the incidence of FHB in the field. This suggests that VDAL likely suppresses pathogen infection by activating host immune responses. Regarding the reduction in DON content, the observed decrease in disease severity may be one contributing factor. However, it is noteworthy that our transcriptomic and qRT-PCR analyses indicate a significant downregulation of specific TRI genes, particularly TRI9 and TRI12, during infection following VDAL application compared with the untreated control. This downregulation may also partially contribute to the reduction in mycotoxin content, although the precise mechanisms underlying the suppression of these genes remain to be elucidated. Our qPCR and DON data were based on three biological replicates, a conventional number. However, given the inherent variability of field-grown samples, future studies with larger sample sizes (e.g., five or six replicates) would further enhance statistical confidence.

In our 2023 field trial, a combined treatment of VDAL with a reduced dose of tebuconazole (90 mL/ha, applied only at the final spray) was included as an exploratory observation. This treatment achieved a control efficacy (69.43%) comparable to VDAL alone (67.00%), indicating no significant additive effect under the tested conditions. Future systematic synergy studies (e.g., checkerboard assays under controlled conditions or dose-response field trials) would be valuable to evaluate potential interactions between VDAL and fungicides more rigorously.

VDAL promotes growth, metabolic traits and physical barriers under nonpathogenic conditions

A pivotal finding of our study is the stark contrast in VDAL’s action in the absence versus presence of the pathogen. Under non-pathogenic conditions, VDAL treatment was associated with a transcriptome profile indicative of enhanced metabolic capacity and basal defense. The upregulation of starch and sucrose metabolism, coupled with photosynthesis-antenna protein, suggests a promotion of photosynthetic efficiency and carbohydrate accumulation in wheat and supporting spike development and grain filling. Concurrently, the enrichment of galactose metabolism and the cutin/suberin/wax biosynthesis pathways suggests improved cell wall integrity and water retention, which promote physical barriers and drought resistance. [41] Furthermore, the induction of alkaloids, flavonoids, and terpenoids biosynthesis pathways underscore VDAL’s role in enhancing chemical defense through antioxidant and antimicrobial compounds (Fig. 2C). [42] The upregulation of cutin, suberin, and wax biosynthesis genes observed at five days post-application suggests that VDAL induces the gradual formation of protective surface layers. The persistent activation of flavonoid-, terpenoid- and alkaloid-related pathways implies VDAL’s sustained chemical defense potential. Rather than triggering immediate defense signaling, VDAL appears to enhance the plant’s capacity for extended stress resilience by reinforcing both physical protection and the metabolic capacity for defense. Nevertheless, our transcriptomic analysis was conducted at a single time point (5 dpi). While this captures late-stage defense responses, future time-course experiments (e.g., 6–48 h post-inoculation) would provide a more complete picture of the early dynamics of VDAL-induced immune activation.

VDAL activates a broad-spectrum immune response upon pathogen infection

While VDAL enhances physical and metabolic traits under pathogen-free conditions, its functional role shifts markedly during infection. In the presence of F. graminearum, VDAL launched a multifaceted defense response (Fig. 9). The specific upregulation of genes involved in SA, JA, and ET signaling; chitin recognition; MAPK cascades; ROS production; calcium signaling; cell wall proteins; and fungal resistance; and other defense-related genes (Figs. 4A and 5). Additionally, stress-responsive pathways such as those associated with high light intensity and heat stress were activated (Fig. 9). Collectively, these findings probably contribute to the establishment of broad-spectrum immunity against pathogen invasion. This study, for the first time, demonstrates that VDAL exerts differential regulatory effects depending on the presence or absence of pathogens.

Fig. 9.

Model illustrating the possible mechanism induced by VDAL and F. graminearum. On the left, VDAL activates signaling pathways associated with seed nutrient accumulation and plant physical and chemical defense mechanisms. On the right, under F. graminearum infection conditions, VDAL significantly activates various hormone signaling (salicylic acid, jasmonic acid, ethylene), ROS and calcium signals related to immune responses, pathogen infection-related signaling pathways, as well as environmental stress-related signaling pathways

The induction of ROS-responsive transcription factor like ZAT12, [43] acyl-CoA oxidase (ACX1), [44] and alpha-dioxygenase (DOX1), [45] implies the critical role of the ROS-mediated signaling in VDAL-mediated defense. Concurrently, the upregulation of the calcium-dependent protein kinase CDPK9 and calmodulin-like (CMLs) underscores the importance of calcium flux in transducing defense signals. The coordinated increase in genes encoding the cell wall protein GP1 further supports active cell wall remodeling as a physical barrier against pathogen invasion. These genes related to ROS bursts, calcium signaling, and structural reinforcement are crucial for effective plant immunity by VDAL treatment and F. graminearum infection.

VDAL coordinates hormone crosstalk through transcriptional regulation to balance defense and growth

Upon F. graminearum infection, VDAL the specific upregulation of key TFs (WRKY70, TIFY11, MYB44, ERF4, ERF54, and RAP2-3) provides insight into the regulatory backbone of VDAL-induced immunity. WRKY70 functions at the convergence point of SA and JA signaling, often serving as a marker for SA-mediated SAR. [21] MYB44, a member of the MYB subfamily 22, is induced by ABA, SA, and JA and regulates WRKY70 expression, thereby modulating the antagonistic interaction between SA and JA signaling. [46] MYB44 is known to activate EIN2, a central regulator of ethylene signaling, which governs both plant development and pattern-triggered immunity (PTI). [47, 48] Therefore, VDAL-induced MYB44 and WRKY70 coordinate SA/JA crosstalk, as well as reinforce SA signaling and PTI, promoting resistance against the hemibiotrophic F. graminearum. Interestingly, the significant induction of TIFY11 [49]—a key negative regulator of JA signaling—suggesting a crucial role of VDAL in fine-tuning the growth-defense balance during the critical grain-filling stage. Under low JA conditions, TIFY11 protein binds to the core transcription factor MYC2, suppressing the expression of JA-responsive genes. Given that JA-mediated defense mechanisms (e.g., insect resistance, antinecrotic trophic pathogens) consume many resources and can inhibit plant growth when constitutively activated. [22] Therefore, the significant induction of TIFY11 likely reflects its role during wheat flowering and filling, when nutrient demand is high and defense suppression helps prioritize growth. Furthermore, TIFY11 has been associated with improved drought tolerance, suggesting that it may also support the adaptation of wheat to heat and water stress during late reproductive phases.

ERFs play pivotal roles in modulating stress responses and defense signaling. The co-regulation of ERFs further integrates ethylene signaling into this defense network. The coordinated upregulation of WRKY70 (SA-related), MYB44, TIFY11 (JA signaling), and ERFs (ethylene pathway) suggests that VDAL enables wheat to balance hormone signals, growth and immunity when facing complex conditions such as ongoing pathogen pressure, the nutrient-demanding grain-filling stage, and heat stress.

VDAL may promote SA accumulation and the establishment of systemic acquired resistance

SA is an essential signaling molecule for the establishment of SAR. [21]Although SA levels were not quantified in this study, the upregulation of SABP2 (SA-binding protein), WRKY70, and the downstream marker gene PR1 (LOC123186194), along with other SAR-associated genes like PAL (phenylalanine ammonia-lyase, LOC123113082), chitin recognition factor (LOC123043226), and APX1 (ascorbate peroxidase 1, LOC123097814), strongly suggests that VDAL may trigger SA -associated signaling, leading to the establishment of SAR. This provides a plausible explanation for the prolonged systemic immunity in the field conditions. Moreover, SA is known to inhibit DON biosynthesis in F. graminearum, [50] SA signaling pathway activation probably explain the observed changes of toxin biosynthesis-related gene expression, though direct causal evidence is lacking.

Although pathogenesis-related genes such as PR1 are commonly used as markers for SA-mediated responses in wheat, it is important to note that in monocots, including wheat and rice, some PR1 gene variants can also be induced by JA. [51, 52] This differs from the hormone signaling mechanisms observed in Arabidopsis and implies the existence of crosstalk between the SA and JA signaling pathways in monocot plants. [26] Therefore, future studies involving direct measurement of SA and JA levels would be valuable for precisely delineating the contribution of each hormone pathway to VDAL-induced resistance in wheat.

VDAL upregulates expression of genes associated with fungal resistance

In addition to activating early defense signaling, VDAL upregulated genes with direct role in fungal resistance, suggesting its broad-spectrum role in enhancing wheat immunity. BAG6 enhances basal immunity and resistance to Botrytis cinerea, whereas UGT13248, a UDP-glucosyltransferase, confers type II resistance by preventing the spread of F. graminearum between spikelets. [53, 54] The indution of BAG6 and UGT13248 by VDAL is likely to contribute to enhanced basal resistance against F. graminearum. The upregulation of the ubiquitin ligase genes (ATL6, RDUF1), [55, 56] known to link to PTI, provides further evidence that VDAL triggers early immune perception. Notably, the significant upregulation of PUB26 is intriguing, given VDAL’s documented interaction with PUB25/26 in Arabidopsis to stabilize MYB6 and enhancing resistance to Verticillium wilt. [29] This suggests a potential conserved mechanism in wheat worthy of further exploration.

VDAL actives heat shock proteins, and stress-responsive genes confer abiotic stress during infection

The impact of VDAL on stress tolerance is not limited to pathogen resistance. It also induces the expression of heat shock proteins (Hsp20, Hsp70, Hsp90, and HSA32) during F. graminearum infection, which are known to improve plant tolerance to high temperatures by maintaining protein homeostasis. [57, 58, 59] Moreover, stress-responsive genes such as ERD7, NOD19, and ClpB162 were also upregulated, highlighting VDAL’s broad role in improving wheat adaptability to various environmental stresses and extending its protective effects beyond pathogen resistance.

Conclusion

This study demonstrated that the plant immune inducer VDAL effectively enhances wheat resistance to F. graminearum not by directly inhibiting fungal growth but by activating host defense responses. Field and transcriptomic evidence revealed that VDAL application significantly reduced FHB severity and DON accumulation. Importantly, VDAL regulates distinct regulatory mechanisms under pathogen-free and infection conditions. In the absence of pathogens, VDAL upregulates pathways associated with photosynthesis, starch biosynthesis, cutin/suberin/wax biosynthesis, and secondary metabolites, thereby promoting wheat development and physicochemical defense. Upon pathogen challenge, VDAL selectively activated immune-related pathways, including SA signaling, reactive oxygen species (ROS) generation, calcium signaling, and cell wall fortification, thereby initiating strong defense responses against F. graminearum. Notably, these immune responses were sustained for at least five days post-application, as evidenced by persistent upregulation of defense-associated genes (e.g., WRKYs, TIFYs, MYB44, and ERFs). This prolonged defense may result from VDAL-triggered systemic acquired resistance (SAR) and/or the ongoing recognition of pathogen-derived molecular patterns during active infection. Together, these findings highlight the dual role of VDAL in activating basal immunity and sustaining long-term defense activation, offering a promising strategy for the integrated management of Fusarium head blight in wheat.

Authors’ contributions

Shulin Cao : Writing-original draft, Visualization, Formal analysis. Ping Lu : Visualization, Formal analysis, Conceptualization. Fuqiang Zhang and Haiyan Sun : Visualization, Software, Formal analysis. Jianguang Xue and Yuanyu Deng : Methodology, Software, Data curation. Xin Zhang and Ling Lin : Visualization, Validation, Software. Wei Li and Huaigu Chen : Writing – review & editing, Funding acquisition.

Funding

This research was funded by the China Agricultural Research System (CAR-03-35), the Jiangsu Agricultural Science and Technology Innovation Fund (CX(23)3010), and the Open Project Program of the State Key Laboratory for Crop Stress Resistance and High-Efficiency Production (SKLCSRHPKF03).

Data availability

The datasets generated during the present study are available from NCBI under BioProject accession number PRJNA1181034. The genomic sequence for the *F. graminearum* strain PH-1 and *T. aestivum* is available at NCBI RefSeq assembly [GCF_018294505.1], and Ensembl Fungi [GCA_900044135.1]. VDAL Sequence data from this article can be found in the GenBank under the accession number: AAS45249.1. Other data generated or analysed during this study are included in this published article and its supplementary information files.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.