# The Fungicidal Effect of Essential Oils of Fennel and Hops against Fusarium Disease of Pea
## 3 Results and discussion
### Determination of Antifungal Activity Ex Vitro
#### 3.2.2 Microscopic Analysis  
The results of microscopic analysis confirmed the effectiveness of HEO and FEO in reducing Fusarium wilt in pea plants. An analysis of the root-stem transition zone showed that the plant tissue was less penetrated by _F. culmorum_ mycelia in seedlings treated with HEO and FEO than in control plants that were not sprayed with essential oils and irrigated with a spore suspension of _F. culmorum_ (Figure 4). Figure 3: Linear regression analysis between symptoms of fusarium wilt and results of qPCR quantification of _TRI-5_ gene (R significant at _p_-0.05). Figure 2: The qPCR quantification of gDNA _Fusarium culmorum_ (_TRI5_ gene) in pea plants treated with different concentrations of essential oils from fennel and hops (pg DNA), (A,B,C,D,E—significant at _p_-0.01 by [PERSON]’s HSD test). An anatomical analysis (Figures 5 and 6) revealed significantly fewer fungal mycelia in plants sprayed with HEO and FEO (Figures 5C-F, 6C-F and 6C-F) than in control plants not treated with essential oils (Figures 5B,B' and 6B,B'). The severity of pathogenic changes on the surface of roots (Figures 4 and 5) and stems (Figures 4 and 6) was influenced by the applied oil and its concentration. Fennel essential oil was more effective than HEO in reducing the symptoms of infection (Figures 4-6), which was confirmed by the disease index of Fusarium wilt. The anatomical analysis of stems demonstrated that FEO was a more potent antifungal agent than HEO (Figure 6). Analysis of root (Figures 4 and 5) and stem (Figure 4) surfaces and anatomical analysis of stems (Figure 6) confirmed that HEO at a concentration of 2000 ppm strongly inhibited mycelial growth of _F. culmorum_. The most severe symptoms of fungal infection, including visible mycelia and brown discolouration indicative of tissue damage, were observed in the epidermis, cortex and vascular bundles of the stem (Figure 6C, arrows) in plants treated with 500 ppm HEO. In plants sprayed with 2000 ppm HEO, symptoms of fungal infection were only observed in the epidermis and peripheral vascular bundles (Figure 6D, arrows). The higher concentration of FEO had a similar antifungal effect. Mycelial growth was more effectively inhibited when FEO was applied at a concentration of 2000 ppm than at a concentration of 500 ppm (Figure 6E,F). Although infection symptoms were similar on the surface of the plant tissue (Figure 6E,F), cross-sections of the stems showed infected vascular bundles in plants treated with FEO at a concentration of 500 ppm (Figure 6E, arrow), whereas in plants sprayed with 2000 ppm FEO, symptoms of fungal infection were observed only on the surface of the stems (Figure 6F, arrows). The direct effects of essential oils on the structure of fungal mycelia have been widely reported in the literature [17; 25; 26], while their contribution to reducing the severity of plant diseases has never been confirmed in anatomical analyses of plant tissues. Essential oils alter the texture, colour and sporulation properties of fungal mycelia [61]. The disadvantage of essential oils is their volatility, which can lead to a loss of their efficacy when used under field conditions [83]. The widespread use in agriculture is opposed by the fact that the approval and registration procedures are very costly, as the evaluation of toxicity and environmental suitability is associated with high costs [84; 85]. The number of registered biological control agents based on EO is significantly lower in Europe than in the United  
Figure 4: Transition region between stem and root of the pea plants: non-treated (**A**) and treated with _Fusarium culmorum_ (**B**), treated _Fusarium culmorum_ with HEO (500 ppm—(**C**), 2000 ppm—(**D**)) and FEO (500 ppm—(**E**), 2000 ppm—(**F**)). Scale bar: 1 and 2 mm. States [86]. In Europe, an increasing number of Eos have been approved as biocides for use in agriculture in recent years. Current research shows the potential for using FEO and HEO to protect peas against _Fusarium_ fungi that cause root necrosis, stem base and Fusarium will.