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%.
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 identified (Table 3). The major components in lavender EO included;Linalool(31.1%),Linalyl anthranilate(16.8%),Benzyl acetate(12.9%), and1,8-Cineole(10.1%). Other components were identified in intermediate proportions includingα-Terpineol acetate (4.92%), ɣ-Terpineol (3.89%), α-Terpineol (3.15%), α-Pinene (2.85%),Dihydrocarveol(2.34%),Dihydromyrcenol(1.81%), andLimonene (1.04%), while, the rest components were present in minor ratios.
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. 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 significant difference in the expression level of the 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. 5. 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 which treated with lavender EO or not at 3 and 6 dpe. In the first 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, pre- and 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 significantly 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 untreated-infected 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 (6). Data obtained indicated that infection of sorghum plants with F. solaniled to an induction in the activities of all studied enzymes when compared with the untreated control plants at 30 and 45 dap. In general, activity of CAT and SOD in sorghum plants at 30 dap was higher than that at 45 dap, while, activity of APX and PPO elevated from 30 to 45 dap. Treating of sorghum plants with lavender EO at different concentrations significantly triggered activity of all testes enzymes, compared 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 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 (7). Results of biochemical analyses of sorghum plants showed that infection with F. solaniled to significantly elevations in the lipid peroxidation, total phenolic and flavonoid 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 significantly 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 flavonoid contents, the obtained data showed that treating of sorghum plants with lavender EO at different concentrations significantly 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