Essential Oil from Lavandula Angustifolia Elicits Expression of Three SbWRKY Transcription Factors Against Sorghum Damping-off and Induces Ultrastructural Alterations in Fusarium Solani

Sorghum damping-off, caused by Fusarium solani (Mart.) Sacc., is a momentous disease which causes economic loss in sorghum production. In this study, antagonistic activity of lavender essential oil (EO) against F. solani was studied in vitro. Their effects on regulation of three transcription factors SbWRKY, the response factor JERF3 and eight defense-related genes, which mediate different signaling pathways, in sorghum were investigated. Their application under greenhouse conditions was also evaluated. The obtained results showed that lavender EO possessed potent antifungal activity against F. solani. Gas chromatography-mass spectrometric analysis revealed that their antifungal activity is mainly attributed to linalyl anthranilate, α-terpineol, eucalyptol, α-Pinene, and limonene. Observations using transmission electron microscope exhibited many abnormalities in the fungal ultrastructures as a response to treating with lavender EO indicating that multi-mechanisms were contributed to their antagonistic behavior. Results obtained from the Real-time PCR investigation demonstrated that all studied genes were overexpressed, at varying extents, in response to lavender EO, however, SbWRKY1 was the highest expressed gene followed by JERF3, which suggest their probable 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 the infection signicantly reduced the disease severity. Moreover, the evaluated growth parameters, activities of the antioxidant enzymes, and total phenolic and avonoid contents were enhanced. In contrast, lipid peroxidation was highly reduced. Results obtained from this study results support the high possibility of using lavender EO for control of sorghum damping-off, however, the eld evaluation was highly needed prior to the usage recommendation. Antifungal activity of EOs and extracts from different which exhibits (glucan binding and glucanase activities), xylanase inhibitor activity, and pathogen recognition (binding with the pathogen cell surface) 42 . The two-genes-clustering (PR2-PR5) 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 revealed the implication of SA-signaling pathway in sorghum resistance against F. solani. Data of 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 dense response 41 . The two-genes-clustering (PR1-PR3) obtained 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 signicant roles in plant resistance against wide range of phytopathogenic fungi 43 . Overexpression of these PR genes revealed the implication of 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 avonoids, lignins, and chlorogenic acid 44 . GST1 encodes the antioxidant-defense enzyme, which involved in the detoxication function against xenobiotics through binding with glutathione 45 . In addition to PR genes, PAL1 and GST1 are also defense genes, which regulated by WRKY transcription factors. The overexpression of all studied genes was supported with the elevated activities of the estimated antioxidant enzymes and total phenol isolated from in 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 15, obtained from the Central Administration for Seed Certication, Egypt, were used in the greenhouse experiment. Shrubs of lavender were obtained


Introduction
Sorghum (Sorghum bicolor (L.) Moench) is among the most important cereal crops worldwide as a source of food/feed, and ethanol production. It is ranked the fth main grain crop with a total global production of more than 59 million tons 1 .
Sorghum damping-off, caused by Fusarium solani (Mart.) Sacc., is a momentous disease which causes seeds and seedlings decay resulting in a signi cant economic loss in the crop yield 2 . In addition, F. solani is a toxigenic fungus which produces dangerous mycotoxins such as trichothecenes and fusaric acid affecting human and animal health 3,4 . The appeared symptoms include seeds decay, discoloration and rotting of the radicles which prevent germination and emergence, and formation of red lesions on the roots of the emerged seedlings which halt their development especially at low temperatures 5 . Various chemical fungicides are available for control of damping-off disease such as Mancozeb, Rizolex, and Benomyl 6 , but the improper use of chemical fungicides is unfavorable owing to their deleterious health effects, and environmental risks 7 .
Essential oils (EOs) and plant extracts have been extensively studied by many researchers as alternatives to chemical fungicides due to their ecological safety, and potent antifungal activities against several phytopathogenic fungi 8- 10 . In this regard, Ghoneem et al. 11 reported a full suppression in the fungal growth of Sclerotinia sclerotiorum by clove essential oil at 2%. The wealthy content of different bioactive components in EOs such as phenols, coumarins, quinines, avonoids, tannins, and fatty acids provides multifunctional and synergistic antifungal potentialities against plant pathogenic fungi. In addition, these multifunctional bioactive compounds guarantee a di culty of microbial-resistance formation through using diverse antagonistic modes of action 9,12 . Moreover, EOs of some medicinal plants may act as elicitors triggering the plant defense-responses against attacking pathogens 8, 13 .
WRKY proteins represent a pivotal plant family of transcription factors (TF) which work via interconnected signaling networks to synchronously regulate a diverse set of defense-responses against biotic and abiotic stresses, as well as metabolic responses 14 . Many investigations have implicated WRKY TFs in regulation of defense-responses against different fungal diseases 15,16 . Overexpression of OsWRKY45 in rice provides a high plant resistance against the blast fungus (Magnaporthe oryzae) via triggering salicylic acid (SA)-signaling pathway genes 17 . While, overexpression of VvWRKY1 in grapevine elicits expression of jasmonic acid (JA)-signaling pathway genes against downy mildew fungus (Plasmopara viticola) 18 . Recently, ninety four SbWRKY TFs were identi ed in sorghum and classi ed into three groups according to their binding domains and type of zinc-nger motifs 19 .
Lavandula angustifolia Mill., frequently known as English lavender, is a owering shrub which belongs to family Lamiaceae. It has many uses as avoring agent in foods, pharmaceutical uses such as soap, perfumes and cosmetics manufactures, as well as many therapeutic applications owing to their antimicrobial, antioxidant, anxiolytic, antispasmodic, and aphrodisiac properties 20 . The present study aimed to 1) investigate the antagonistic activity of lavender EO against F. solani in vitro, especially their ultra-structures, 2) study their effect(s) on regulation of three SbWRKY TFs (1, 19, and 45), Jasmonate and ethylene-response factor 3 (JERF3) and eight defense-related genes, which mediate SA, JA and ethylene (ET)-signaling pathways, in sorghum against Fusarium damping-off, 3) evaluate their biocontrol activity under greenhouse conditions, as well as their effects on the growth and biochemical plant parameters.

Results
Screening for antifungal activity of lavender EO in vitro Antifungal activity of lavender EO was assessed in vitro against F. solaniat 0, 0.5, 0.75, 1, 1.25, and 1.5 % (Fig. 1). The mean reductions in the fungal growth are presented in Table 2. All tested concentrations exhibited inhibitory potentiality in varying extents compared with the control treatment. The growth inhibition elevated with the increment in the concentration of lavender EO. The highest growth inhibition (97.6%) was obtained at 1.5 % recording 2 mm radial growth compared with the control. While, the lowest growth inhibition was recorded at the concentration of 0.5%.

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TEM Observations of TEM for untreated (control) hyphae of F. solani exhibited normal ultrastructure. Thin cell wall and plasmalemma embracing the cytoplasm with electron-lucent lipid globules, nucleus, and vacuoles were noted (Fig. 2 a and b). In contrast, TEM observations of F. solani hyphae treated with lavender EO showed considerable ultrastructural alterations. Thick cell wall and plasmalemma enclosing an electron-dense cytoplasm were observed. Big vacuoles containing electron-dense materials, and absence of the lipid globules were also noted ( Fig. 2 c and d).

GC-MS
The chemical composition of lavender EO was analyzed via GC-MS system (Fig. 3). Twenty-eight compounds in varying proportions were identi ed ( Transcript levels of three SbWRKY TFs and nine defenserelated genes. Transcriptional expression pro les of three SbWRKY TFs, JERF3 and eight defense-related genes in sorghum shoot were studied 3 and 6 days post emergence (dpe) (Fig. 4). Of all studied genes, SbWRKY1was the highest expressed gene followed by JERF3. For SbWRKY1expression, infection of sorghum plants with F. solanior 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 that in the EO treated-plants when compared with the untreated control plants. However, the highest expression level was recorded for the infected plants, which treated with lavender EO (43-fold at 3 dpe). For all treatments, the expression level of SbWRKY1at 6 dpe was lower than that at 3 dpe. The expression level of JERF3 came in second after SbWRKY1and was triggered by infection with F. solaniand/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, SbWRKY19and SbWRKY45,infection with F. solaniand/or treating with lavender EO induced the gene expression level at 3 and 6 dpe in varying degrees. However, the highest expression was observed for the infected plants which treated with lavender EO, followed by the untreated-infected plants, while, treatment of lavender EO came in third, when compared with the untreated control plants. Regarding PAL1, AFPRT, and GST1, the untreated-infected sorghum plants or infected plants which treated with EO showed considerable up-regulation in the transcript level of the three genes, but the dual treatment was more inducer than the infection treatment. In contrast, sorghum plants treated with lavender EO did not exhibit any signi cant difference in the expression level of the three genes, when compared with the untreated control plants. In all expression pro les, 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. 5. As seen from the heat map, all tested treatments are grouped into two main clusters, the rst represents the untreated control plants, and the lavender-EO-treated plants at 3 and 6 dpe, while the other represents the infected plants which treated with lavender EO or not at 3 and 6 dpe. In the rst cluster, the untreated control plants at both investigated times (3 and 6 dpe) are grouped together in a separate subcluster, while, the lavender-EO-treated plants at the same times are grouped together in the other subcluster. In the second main cluster, the infected plants at the investigated times are grouped together in a separate subcluster, while, the infected plants which treated with lavender EO at 3 and 6 dpe are grouped together in another separate subcluster. Concerning the gene clustering, all genes are grouped into two main clusters, the transcription factorSbWRKY45is grouped in a separate out-cluster revealing its unique behavior, while, the other main cluster included all the other investigated genes.
Moreover, the hierarchical clustering heat map shows two-genes-clustering between GST1-PAL1, PR5-PR2, AFPRT-PR12, PR3-PR1, and SbWRKY19-JERF3. In general, the hierarchical clustering expression exhibited high up-regulation of the investigated genes in case of the infection treatments, whether treated with lavender EO or not. The maximum transcription levels were observed for the infected plants, which treated with lavender EO at 3 dpe.

Disease assessment
Disease assessment data of the infected sorghum seedlings in response to treatment with lavender EO at different concentrations are presented in Table (4). The obtained data indicate that the infection with F. solanicaused 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, 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 the increment in the EO concentration. In this regard, the best result was recorded for the sorghum grains, which treated with lavender EO at 1.5% prior to the infection recording 17.7% mortality, when compared with that which treated with the chemical fungicide.
Effect on the plant growth Results of the growth parameters evaluation obtained from the greenhouse experiment in response to treatment with lavender EO at different concentrations and infection with F. solani are presented in Table (5). Infection of sorghum plants with F. solaniled to a considerable reduction in the evaluated growth parameters at 30 and 45 dap, when compared with the untreated control plants. In contrast, treating of sorghum grains with lavender EO signi cantly enhanced the growth of sorghum plants compared with the untreated control plants. The growth promoting effect elevated with the increment in the EO concentration. The highest growth parameters were recorded for the sorghum plants treated with lavender EO at 1.5% at both harvests 30 and 45 dap. Compared to the treatment with the chemical fungicide, sorghum plants treated with lavender EO prior to infection with F. solanishowed higher growth records, regarding to the plant height, shoot and root dry weights, than that recorded for the untreatedinfected sorghum plants. In this regard, the growth enhancing effect is directly proportional to the EO concentration at 30 and 45 dap.

Effects on activities of antioxidant enzymes
Effects of lavender EO on activities of different antioxidant enzymes of sorghum plants infected with F. solani are shown in Table ( with the untreated control. However, the inducing effect resulted by the infection was more than that of the lavender EO treatments at both studied times. For all studied enzymes, the highest enzyme activity was recorded for the infected sorghum plants that treated with lavender EO at 1.5%, when compared with the treatment of the chemical fungicide. In this regard, the inducing effect on enzymes activity is directly proportional to the EO concentration at 30 and 45 dap.

Effects on lipid peroxidation, total phenolic and avonoid contents
Effects of lavender EO on lipid peroxidation, total phenolic and avonoid contents of sorghum plants infected with F. solani are presented in Table (7). Results of biochemical analyses of sorghum plants showed that infection with F. solaniled to signi cantly elevations in the lipid peroxidation, total phenolic and avonoid contents at 30 and 45 dap, when compared with the untreated control plants. In contrast, treating with lavender EO at different concentrations did not affect lipid peroxidation of sorghum plants.
Whilst, treating of the infected sorghum plants with lavender EO signi cantly reduced the lipid peroxidation, compared with the treatment of the chemical fungicide. This reducing effect is directly proportional to the EO concentration at 30 and 45 dap. The lipid peroxidation in sorghum plants at 30 dap was higher than that at 45 dap. Regarding to the total phenolic and avonoid contents, the obtained data showed that treating of sorghum plants with lavender EO at different concentrations signi cantly induced both parameters in a direct proportional relationship at 30 and 45 dap. The highest contents were recorded for the infected sorghum plants treated with lavender EO at 1.5%, compared with the chemical fungicide treatment at 30 and 45 dap. In general, both contents in all sorghum plants treatments elevated from 30 to 45 dap

Discussion
The present work aimed to evaluate lavender EO in response 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 obtained results indicated that lavender EO possesses antifungal activity at different concentrations against F. solani. This result is in agreement with ndings reported by Bahmani and Schmidt 21 , 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, avonoids, 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 wall and plasma membrane, suppression of metabolic enzymes, and DNA damage 23 . 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. Majority of the antifungal activity of lavender EO is attributed to linalool, their most abundant bioactive component. Recent researches have reported a potent antifungal activity for linalool 24 . Their fungitoxic effect can be explained in the light of interference with cell wall biosynthesis and disrupting permeability of plasmalemma 25 . In addition, α-terpineol has been reported also as a potent antifungal agent, their antimycotic effect was suggested to be due to its activity on cytoplasmic degeneration and hyphal distortions 26 . These antifungal modes of action were con rmed by our TEM observations. In this regard, the TEM observations revealed many abnormalities in ultrastructures of F. solani treated with lavender EO indicating that multi-mechanisms were contributed to their antagonistic behavior such as thickening of cell wall and plasmalemma, which con rm mechanism of interfering with their integrity, resulting in loss of their permeability. Thickening of the cell wall and plasmalemma leads to restriction of the cellular exchange of ions and molecules with the surrounding medium, which nally results in the cell death 8 . In addition, another antifungal mechanism was observed by TEM, which is the cytoplasmic coagulation. This effect is correlated with the impairment of the plasmalemma, which followed by condensation and coagulation of the cytoplasm and nally cell death 27 . Absence of lipid globules in the treated F. solani hyphae was another TEM note. Lipid droplets play important roles in the fungal cell as energy reserves, preventing lipotoxicity, and regulating some physiological processes 28 . 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 their 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 encountered stresses, plants initiate some 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 29 . Transcription factors, such as WRKY proteins, play important roles in synchronously organizing the transcriptionregulatory-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 plant. The W-boxes are exist in clusters suggesting coordinated interactions of several WRKY TFs 30 . 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) 31 . 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 32,33 . 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 34 . In this concern, Zhang et al. 35 reported the involvement of ERF3 in triggering an array of defense responses against Blumeria graminis in wheat at early stages via 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 of all the studied genes revealing its unique behavior. This result is in agreement with that obtained by Shimono et al. 36 who reported the vital role of OsWRKY45 in triggering plant resistance against blast fungus (Magnaporthe grisea) on rice. The same concept was reported by Qiu and Yu 37 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 con rmed 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 38 . It is well 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 39 . 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 has 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) 40 . 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 41 . 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) 42 . The two-genes-clustering (PR2-PR5) 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 revealed the implication of SA-signaling pathway in sorghum resistance against F. solani. Data of 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 dense response 41 . The two-genes-clustering (PR1-PR3) obtained 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 signi cant roles in plant resistance against wide range of phytopathogenic fungi 43 . Overexpression of these PR genes revealed the implication of 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 avonoids, lignins, and chlorogenic acid 44 . GST1 encodes the antioxidant-defense enzyme, which involved in the detoxi cation function against xenobiotics through binding with glutathione 45 . In addition to PR genes, PAL1 and GST1 are also defense genes, which regulated by WRKY transcription factors. The overexpression of all studied 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.

Materials And Methods
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 15, obtained from the Central Administration for Seed Certi cation, Egypt, were used in the greenhouse experiment. Shrubs of lavender were obtained from the Ornamental Plants Cultivation Station, Agriculture Research Center, Egypt.

Essential oil extraction
Lavender EO was extracted from 200 g of air-dried lavender owers via hydro-distillation for 60 min using Clevenger apparatus as described by Zheljazkov et al. 46 . The EO was then ltered and stored in dark bottle at 4°C until use.

Screening for antifungal activity of lavender EO in vitro
Antifungal activity of lavender EO was assessed against mycelial growth of F. solani in vitro using agar plate technique. PDA plates supplemented with lavender EO at nal concentrations of 0.5, 0.75, 1.0, 1.25, and 1.5% were used. The tested concentrations were prepared by adding suitable volumes of EO to 100 mL Erlenmeyer asks containing melted PDA medium before solidi cation and 0.5 % Tween-80. 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.

Transmission electron microscopy (TEM)
To investigate effects of lavender EO on ultrastructure of F. solani using TEM, samples of the treated plates were xed 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. 47 . 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).
The EO sample was injected at ow 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 volatage 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) lled 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 singly 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 kept under greenhouse conditions (27/21°C day/night temperature, 65% humidity). The applied treatments were designated as follows: C: untreated control, EO 1.0, EO 1.25, EO 1.5 : treated with lavender EO at 1.0, 1.25, and 1.5% respectively, P: infected with F. solani, P + EO 1.0 , P + EO 1.25 , and P + EO 1.5 : infected with F. solani and treated with lavender EO at 1.0, 1.25, and 1.5% respectively, and P + F: infected with F. solani and treated with chemical fungicide. All pots were evaluated for damping-off incidence 45-days after planting (dap). Percentages of seed rot, pre-and post-emergence damping-off were recorded.
Quantitative Real-Time PCR (qRT-PCR) Total RNA extraction and cDNA synthesis.
Total RNA was extracted from fresh sorghum shoot at 3 and 6 days post seedling emergence (dpe) using RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. The extracted RNA was incubated with DNase for 1 h at 37°C and quanti ed using a NanoDrop 1000 spectrophotometer (Thermo Scienti c, USA).

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 βactine gene was used as a reference gene. Sequences of used primers are presented in Table (1). 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 sec, 58°C for 25 sec and 72°C for 30 sec). 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 and Schmittgen 48 .

Evaluation of plant growth
For each treatment, three plants were carefully uprooted 30 and 45 dap, washed with tap water to remove soil particles, and evaluated for plant height, shoot and root dry weights. Dry weights were measured after the samples oven-dried at 80°C for 48 h.

Biochemical analyses
Preparation of crude plant extract Samples of plant roots (2g) were ground and homogenized in 5 mL of 100 mM phosphate buffer (pH 7). The homogenate was centrifuged at 15000 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 49 . Assay of enzymes activities

Statistical analyses
Statistical signi cances were analyzed using the software CoStat (version 6.4). Comparisons between the means were performed using Duncan's multiple range test 57 at P ≤ 0.05. The hierarchical clustering analysis was performed using BioVinci Software (Bioturing, San Diego, CA, USA).

Declarations
Authors con rm that all the methods and experiments were carried out in accordance with relevant guidelines and regulations.

Funding
This research did not receive speci c grants from funding agencies in the public, commercial, or not-forpro t sectors.

Con icts of Interest
The authors declare no competing interests ( nancial and non-nancial interests).
https://doi.org /10.1006/abio.1976.9999 (1976 Tables   Table 1 Sequences of primer used in this study.  Table 2 Growth inhibition (%) of Fusarium solani when exposed to lavender essential oil at different concentrations*.  Table 3 Chemical composition of lavender essential oil using GC-MS system.     Figure 1 Antifungal activity of lavender essential oil at different concentrations against Fusarium solani in vitro.  Histograms showing relative transcriptional expression levels of three SbWRKY transcription factors and some defense-related genes in sorghum plants infected with Fusarium solani and/or treated with lavender essential oil at 1.5% after 3 and 6 days post-emergence (dpe). Where, C: untreated control, P: infected with F. solani, EO: treated with lavender essential oil at 1.5%, and P+EO: infected with F. solani and treated with lavender essential oil at 1.5%. In each time for each studied gene, columns superscripted with the same letter are not signi cantly different according to Duncan's multiple range test (P≤0.05). Each value represents the mean of three biological replicates; each sample was analyzed in triplicate.
Error bars represent standard errors.

Figure 5
Hierarchical clustering heat map of transcriptional expression of three SbWRKY transcription factors and some defense-related genes in sorghum plant infected with Fusarium solani and/or treated with lavender essential oil at 1.5% after 3 and 6 days post-emergence (dpe). Where, C: untreated control, P: infected with F. solani, EO: treated with lavender essential oil at 1.5%, and P+EO: infected with F. solani and treated with lavender essential oil at 1.5%.