Essential oil from Lavandula angustifolia elicits expression of three SbWRKY transcription factors and defense-related genes against sorghum damping-off

Sorghum damping-off, caused by Fusarium solani (Mart.) Sacc., is a serious disease which causes economic loss in sorghum production. In this study, antagonistic activity of lavender essential oil (EO) at 0.5, 0.75, 1.0, 1.25, 1.5, and 1.6% against F. solani was studied in vitro. Their effects on regulation of three SbWRKY transcription factors, the response factor JERF3 and eight defense-related genes, which mediate different signaling pathways, in sorghum were investigated. Effects of application under greenhouse conditions were also evaluated. The results showed that lavender EO possesses potent antifungal activity against F. solani. A complete inhibition in the fungal growth was recorded for lavender EO at 1.6%. Gas chromatography–mass spectrometric analysis revealed that EO antifungal activity is most likely attributed to linalyl anthranilate, α-terpineol, eucalyptol, α-Pinene, and limonene. Observations using transmission electron microscopy revealed many abnormalities in the ultrastructures of the fungal mycelium as a response to treating with lavender EO, indicating that multi-mechanisms contributed to their antagonistic behavior. Results obtained from Real-time PCR investigations demonstrated that the genes studied were overexpressed, to varying extents in response to lavender EO. However, SbWRKY1 was the highest differentially expressed gene followed by JERF3, which suggest they play primary role(s) in synchronously organizing the transcription-regulatory-networks enhancing the plant resistance. Under greenhouse conditions, treating of sorghum grains with lavender EO at 1.5% prior to infection significantly reduced disease severity. Moreover, the growth parameters evaluated, the activities of antioxidant enzymes, and total phenolic and flavonoid contents were all enhanced. In contrast, lipid peroxidation was highly reduced. Results obtained from this study support the possibility of using lavender EO for control of sorghum damping-off. However, field evaluation is highly needed prior to any usage recommendation.

Transcript levels of three SbWRKY TFs and nine defense-related genes. Transcriptional expression profiles of three SbWRKY TFs, JERF3 and eight defense-related genes in sorghum shoot were studied 3 and 6 days post emergence (dpe) (Fig. 5). In every case, EO in the presence of the pathogen increased the level of expressions as measured by mRNA levels and uninoculated controls had the least mRNA. Inoculated plants in the absence of EO were also higher than controls and in most but not all cases expression induced by EO alone was also above the control level. Of all studied genes, SbWRKY1 was the highest expressed gene followed by JERF3. For SbWRKY1 expression, infection of sorghum plants with F. solani or treating with lavender EO induced their transcript level, but the transcriptional expression in the infected plants was much higher (21-fold at 3 dpe) than in the EO treated-plants when compared with the untreated control plants. However, the highest expression level was recorded for the infected plants also treated with lavender EO (43-fold at 3 dpe). For all treatments, the expression level of SbWRKY1 at 6 dpe was lower than that at 3 dpe. The expression level of JERF3 came in second after SbWRKY1 and was triggered by infection with F. solani and/or treating with lavender EO, compared with the untreated control plants, but the expression level of the dual treatment was higher than the single treatments recording 29-and 28-fold at 3 and 6 dpe, respectively. Concerning PR1, PR2, PR3, PR5, PR12, SbWRKY19 and SbWRKY45, infection with F. solani and/or treating with lavender EO induced the gene expression level at 3 and 6 dpe in varying degrees. Regarding PAL1, AFPRT, and GST1, the untreated-infected sorghum plants or infected plants which were treated with EO showed considerable up-regulation in the transcript level of the three genes, but the dual treatment was more highly induced than the infection-alone treatment. In contrast, sorghum plants treated with lavender EO did not exhibit any significant difference in the expression level of these three genes, when compared with the untreated control plants. In all expression profiles, the transcript level of the studied genes reduced from 3 to 6 dpe.
Hierarchical clustering analysis. Hierarchical clustering heat map of transcriptional expression of the investigated genes in sorghum shoot is illustrated in Fig. 6. As seen from the heat map, all tested treatments are grouped into two main clusters, the first represents the untreated control plants, and the lavender-EO-treated plants at 3 and 6 dpe, while the other represents the infected plants whether treated with lavender EO or not at Table 1. Growth inhibition (%) of Fusarium solani when exposed to lavender essential oil at different concentrations. Values followed by the same letter are not significantly different according to Duncan's multiple range test (P ≤ 0.05), each value represents the mean of 3 replicates ± SD. Chemical fungicide = nystatin at 50 µg/mL. Disease assessment. Disease assessment data of the infected sorghum seedlings in response to treatment with lavender EO at different concentrations are presented in Table 3. The data indicated that the infection with F. solani caused damping-off of sorghum leading to up to 92% mortality, when compared with the untreated control treatment. Typical symptoms of Fusarium damping-off were recorded, including seed rotting, and preand post-emergence damping-off. In contrast, treating of sorghum grains with lavender EO prior to infection with F. solani led to a reduction in the disease severity, which increased with increased EO concentration. In this regard, the best result was recorded for the sorghum grains treated with lavender EO at 1.5% prior to the infection (17.7% mortality), which was essentially identical to treatment with the chemical fungicide.     Effects on lipid peroxidation, total phenolic and flavonoid contents. Effects of lavender EO on lipid peroxidation, total phenolic and flavonoid contents of sorghum plants infected with F. solani are presented in Table 6. Results of biochemical analyses of sorghum plants showed that infection with F. solani led to significant elevations in lipid peroxidation, total phenolics and flavonoids at 30 and 45 dap, when compared with the Table 2. Chemical composition of lavender essential oil using GC-MS system.

Discussion
The present work aimed to evaluate the effect of lavender EO in regard to their antifungal activity against F. solani in vitro, and their resistance-inducing activity against Fusarium damping-off in sorghum, especially on SbWRKY TFs. In vitro, the results indicated that lavender EO possesses concentration dependent antifungal activity against F. solani. This result is in agreement with findings reported by Bahmani and Schmidt 23 , and Behmanesh et al. 22 . Antifungal activity of EOs and extracts from different medicinal plants, including lavender EO, has been reported by many researchers 8,9 . The chemical composition of medicinal plants comprises various bioactive phytochemicals such as coumarins, flavonoids, terpenes, anthocyanins, and tannins, which may contribute to the fungitoxic activity. Different mechanisms have been described in this concern including interfering with permeability and integrity of fungal cell walls and plasma membranes, suppression of metabolic enzymes, and DNA damage 24 . GC-MS analysis of lavender EO showed existence of some bioactive constituents with a known antifungal background including linalool as the main bioactive component, in addition to linalyl anthranilate, α-terpineol, 1,8-cineole (eucalyptol), α-Pinene, and limonene. Most of the antifungal activity of lavender EO is attributed to linalool, simply because it is the most abundant bioactive component. Recent researchers have reported a potent antifungal activity for linalool 25 . It's fungitoxic effect can be explained in the light of interference with cell wall biosynthesis and disrupting permeability of plasmalemma 26 . In addition, α-terpineol has been reported also as www.nature.com/scientificreports/ a potent antifungal agent and its antimycotic effect has been suggested to be due to its activity on cytoplasmic degeneration and hyphal distortions 27 . These antifungal effects were observed in our TEM observations. In this regard, the TEM observations revealed many abnormalities in cellular ultrastructure of F. solani treated with lavender EO such as thickening of cell wall and plasmalemma, indicating that multi-mechanisms contributed to the observed antagonistic behavior, that are compatible with a result loss of permeability. Thickening of the cell wall and plasmalemma leads to restriction of the cellular exchange of ions and molecules with the surrounding medium, which finally results in cell death 8 . In addition, another antifungal mechanism was observed by TEM, which is cytoplasmic coagulation. This effect is correlated with the impairment of the plasmalemma, followed by condensation and coagulation of the cytoplasm and finally cell death 28 . Absence of lipid globules in the treated F. solani hyphae was also observed with TEM. Lipid droplets play important roles in the fungal cell as energy reserves, preventing lipotoxicity, and regulating some physiological processes 29 . Absence of lipid globules reveals that the fungal cell is suffering stress conditions. At the molecular level, twelve genes including three SbWRKY TFs, JERF3 and eight defense-related genes, representing SA-, JA-and ET-signaling pathways, were selected in this study as pathway reporter genes. Transcriptional expression levels of these genes were investigated in sorghum shoot in response to application of lavender EO and/or infection with F. solani at 3 and 6 dpe. The obtained results demonstrated that SbWRKY1 was the highest expressed gene followed by JERF3, which suggest probable primary role(s) in the plant resistance in response to these treatments. Plants are subjected to multiple environmental stresses, including pathogenic fungi, and energetically respond to these challenges to survive. In order to overcome the stresses encountered, plants initiate transcriptional cascades through cellular signaling pathways. These pathways interact in coordination with each other via signaling molecules leading to stimulation of the defensive-gene-regulatory networks 30 . Transcription factors, such as WRKY proteins, play important roles in synchronously organizing the transcription-regulatory-networks enhancing the plant responses against biotic and abiotic stresses 16 . WRKY TFs bind to W-boxes found in the stress-inducible promoters of many defense-related genes in plants. The W-boxes exist in clusters, suggesting coordinated interactions of several WRKY TFs 31 . In this regard, WRKY1 TF has been reported as a key element mediating induced resistance against infection with Alternaria solani in wild tomato (Solanum arcanum) 32 . WRKY1 regulates SA-signaling pathway via interaction with NPR1 gene (Natriuretic Peptide Receptor 1), which functions as a master regulator in the orchestration of the plant-defense-responses, controlling expression of more than 2000 defense-related genes 33,34 . JERF3, which functions as a key element of ET/JA-signaling pathways, activates multiple defense responses via binding to the GCC box located in the promoters of some defense-related genes 35 . In this regard, Zhang et al. 36 reported the involvement of ERF3 in triggering an array of defense responses against Blumeria graminis in wheat at early stages via the SA-signaling pathway, and against F. graminearum or Rhizoctonia cerealis at late stages via ET/JA-signaling pathways. In this study, one of the most interesting results obtained by the hierarchical clustering analysis is the single clustering of SbWRKY45 away from the other studied genes revealing its unique behavior. This result is in agreement with that obtained by Shimono et al. 37 who reported the vital role of OsWRKY45 in triggering plant resistance against the blast fungus (Magnaporthe grisea) on rice. The same concept was reported by Qiu and Yu 38 against Pyricularia oryzae and Xanthomonas oryzae on rice, and in Arabidopsis. The WRKY45-induced resistance included overexpression of some PR genes, particularly, PR1 and PR2 (markers of systemic acquired resistance). In addition, he confirmed the mediation of OsWRKY45 to SA-signaling pathway. Likewise, WRKY19 has been also reported to be involved in induction of plant resistance against powdery mildew of barley 39 . It is known that induction of SA-signaling pathway leads to overexpression of the pathogenesis-related (PR) genes PR1, PR2, and PR5, while, triggering JA-signaling pathway induces PR3, PR4, and PR12 genes 40 . In this regard, data obtained in this study revealed overexpression of PR1 (antifungal), PR2 (β-1,3-glucanase), and PR5 (Thaumatin-like protein) which are SA-responsive defense genes. This result is in accordance with the reported overexpression of WRKY genes. PR1 proteins are highly abundant in plants during biotic-and abiotic-stress responses and have been widely used as a defense marker. Unlike PR2 and PR5 proteins, which have known antifungal enzymatic activities, the antifungal mechanism of PR1 proteins remains unclear. However, recent studies have suggested multiple roles of PR1 proteins including sterol-binding activity, hypersensitivity response (cell death), and harboring an embedded defense-signaling peptide (CAP-Derived Peptide 1) 41 . PR2 encodes the lytic enzyme β-1,3-glucanase, which hydrolyz β-1,3-glycosidic bond in the 1,3-glucan molecules, degrading the cell walls of attacking phytopathogenic fungi 42 . PR5 encodes antifungal protein which exhibits fungal-cell-wall-lytic activity (glucan binding and glucanase activities), xylanase inhibitor activity, and pathogen recognition (binding with the pathogen cell surface) 43 . The two-genes-clustering (PR2 and PR5) with similar patterns obtained in this study can be explained in the light of their shared glucanase activities and the same SA-signaling pathway. Overexpression of these PR genes implicate a role for SA-signaling in sorghum resistance against F. solani. Data from the quantitative Real-Time PCR (qRT-PCR) obtained in this study revealed overexpression of PR3 (chitinase 15), and PR12 (Plant defensin 1), which are JA-responsive defense genes. PR3 encodes the antifungal enzyme chitinase, which catalyze hydrolysis of β-1,4 bonds between N-acetylglucosamine subunits of chitin molecules, the main constituent of the fungal cell wall. PR1 and PR3 proteins synergistically inhibit the fungal growth as a plant defense response 42 . Clustering of PR1 and PR3 observed in this study is in accordance with the synergism reported between the two proteins in the literature. PR12 encodes antifungal and cytotoxic proteins, which have significant roles in plant resistance against wide range of phytopathogenic fungi 44 . Overexpression of these PR genes revealed the participation of the JA-signaling pathway in sorghum resistance against F. solani. PAL1 is the key gene in the phenylpropanoid pathway regulating biosynthesis of an array of antifungal polyphenolic compounds in plant including flavonoids, lignins, and chlorogenic acid 45 . GST1 encodes the antioxidant-defense enzyme, which involved in the detoxification function against xenobiotics through binding with glutathione 46 . In addition to PR genes, PAL1 and GST1 are also defense genes, which regulated by WRKY transcription factors. The overexpression of all studied Scientific Reports | (2022) 12:857 | https://doi.org/10.1038/s41598-022-04903-x www.nature.com/scientificreports/ genes was supported with the elevated activities of the estimated antioxidant enzymes and total phenol content explaining the synergistic effect of lavender EO and infection with F. solani in triggering the sorghum resistance.

Fungal inoculum, sorghum cultivar, and lavender shrubs. A virulent isolate of the fungus F. solani
(GenBank accession no.: KJ831188), isolated from sorghum seedling showing damping-off symptoms, was used in this study. The fungal isolate was maintained on potato dextrose agar (PDA) slants and kept at 4 °C until use. For inoculum preparation, fungal spores from 7-days-old PDA cultures of F. solani were harvested using sterile water and the spore suspension was adjusted at 1 × 10 6 spore mL −1 . Sorghum grains cv. Giza  were used as positive control. Untreated PDA plates were used as negative control. The PDA plates were inoculated in the centers with 7-mm-diameter discs taken from active margins of 7-days-old culture of F. solani. For each treatment, three plates were used. All plates were incubated in dark at 25 ± 2 °C until full fungal growth was obtained in the control plate. Diameter of the fungal colony in each plate was measured and the reduction percentage in mycelial growth was calculated as follows: where C = colony diameter of the control plate, and T = colony diameter of the treated plate.

SEM.
To investigate effects of lavender EO on morphology of F. solani using SEM, one block from F. solani culture on PDA plate and another one block from F. solani culture treated with lavender EO were separately processed using tissue processor (Leica Biosystems, Inc.). The two blocks were fixed using osmium oxide, dehydrated using ethanol, and dried using a critical point drier (EMS 850), and then coated with gold using a sputter coater (EMS 550). Morphology of the fungal structures was observed using SEM (JEOL JSM-6510LV).

TEM.
To investigate effects of lavender EO on ultrastructure of F. solani using TEM, samples of the treated plates were fixed using 3% glutaraldehyde in phosphate buffer at pH 6.8, followed with 1% osmium tetroxide, then dehydrated in a gradual ethanol series as described by Alberto et al. 48 . The dehydrated specimens were embedded in plastic epoxy resin and cut to ultra-thin sections using Reichert ultramicrotome, and stained with uranyl acetate followed by lead citrate. The sections were examined using JEOL-TEM (model JEM-1230).

GC-MS.
Chemical constituents of lavender EO were identified using a GCMS-QP2010 system (Shimadzu, Japan). The EO sample was injected at flow rate of 1.5 mL min −1 via a DB-5 column (60 m × 0.25 mm, 0.25 μm thick) using helium as a carrier at 300 °C. The ion source temperature was 210 °C, while the interface temperature was 300 °C, at an ionization voltage of 70 eV. Retention time and mass spectra were used to identify the EO composition using the NIST11library (Gaithersburg, USA).
Greenhouse experiment. Plastic pots (15 cm diameter) filled with sterile sandy-clay soil (1:2 v/v) were used. For soil infestation, the spore suspension of F. solani (1 × 10 6 spore mL −1 ) was mixed with upper layer soil of the pots at the rate of 2% (v/v) ten days before planting. Sorghum grains were soaked separately in lavender EO at different concentrations (1, 1.25, and 1.5%) for 2 h, then air-dried before planting. For positive control, sorghum grains were treated with the chemical fungicide Rhizolex-T as seed dressing at the recommended dose of 3 g kg −1 grains. For each treatment, ten sorghum grains were sown in each pot. Pots planted with surfacesterilized sorghum grains were used as a control treatment. Four replicates were used for each treatment. All pots were regularly irrigated as required, arranged in a complete randomized design, and kept under greenhouse conditions ( For cDNA synthesis, RT-PCR kit (Qiagen, Germany) was used according to the manufacturer's instructions. The reaction mixture (20µL) contained 2.5 µL dNTPs (2.5 mM), 5 µL 5×-buffer with MgCl 2 , 4 µL oligo (dT) primer (20 pmol µL −1 ), 0.2 µL reverse transcriptase enzyme (Omniscript RT, Qiagen, Germany) and 2 µL RNA. PCR amplification was performed using a thermal cycler (Promega, Germany), at 42 °C for 2 h and 65 °C for 20 min, the cDNA was then stored at − 20 °C until used.
qRT-PCR. The reaction mixture included 1 µL of template, 12.5 µL of SYBR Green Master Mix (Bioline, Germany), 1 µL of forward primer, 1 µL of reverse primer, and sterile RNase free water for a total volume of 20 µL. A β-actine gene was used as a reference gene. Sequences of used primers are presented in Table 7. The real time PCR program was performed using a Rotor-Gene-6000-system (Qiagene, USA) as follows: one cycle at 95 °C for 10 min, 40 cycles (95 °C for 20 s, 58 °C for 25 s and 72 °C for 30 s). For each sample, three biological and three technical replicates were performed. The comparative CT method (2 −ΔΔCT ) was used to analyze the relative mRNA expression levels according to Livak  Biochemical analyses. Preparation of crude plant extract. Samples of plant roots (2 g) were ground and homogenized in 5 mL of 100 mM phosphate buffer (pH 7). The homogenate was centrifuged at 15,000 rpm for 20 min, then the supernatant was collected and used as crude extract for next enzyme assays and biochemical analyses. The protein content was estimated for the assayed enzymes according to Bradford 50 .
Assay of enzymes activities. Activity of CAT was determined according to Aebi 51 . Activity of SOD was determined according to Misra and Fridovich 52 . Activity of ascorbate peroxidase enzyme (APX) was determined according to Nakano and Asada 53 . Activity of PPO was determined according to Duan et al. 54 . Table 7. Sequences of primer used in this study. www.nature.com/scientificreports/ Lipid peroxidation, and total phenolic and flavonoid contents. Lipid peroxidation was measured as described by Heath and Packer 55 . Total phenolic content was estimated according to Slinkard and Singleton 56 . Total flavonoid content was determined as described by Wang et al. 57 .
Statistical analyses. Statistical significances were analyzed using the software CoStat (version 6.4). Comparisons between the means were performed using Duncan's multiple range test 58 at P ≤ 0.05. The hierarchical clustering analysis was performed using BioVinci Software (Bioturing, San Diego, CA, USA) (Supplementary Figure 1).

Ethics declaration.
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