The agar medium used in this study was 3% malt extract agar (MEA) amended with 0.5% mycological peptone, and potato dextrose agar (PDA) (Biokar, France). The monoterpene carvacrol (99%) used in this study was obtained from Flagresso, Austria.

Fusarium oxysporum, F. solani, and F. nivale strains used in the present study were isolated from diseased leaves, stems, and roots of ryegrass and from soil samples before being identified. For morphological characterisation, macroscopic traits such as the colony appearance, colour, pigmentation and growth rate were observed on potato dextrose agar (PDA) according to Leslie & Summerell (2006). Morphological identification was also performed based on the morphological characteristics observed at optical microscope as described by Booth (1971) and Leslie & Summerell (2006).

The morphological identification was further confirmed by DNA sequencing. Single hyphal tip isolates were grown on cellophane overlain on PDA for 10 days. The mycelium was lyophilized and 10 mg of lyophilized mycelium, grinded with 5 mm iron beads. Then, fungal DNA was extracted using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol (Fallahi et al., 2019). The quality and quantity of the DNA obtained were evaluated by measuring the concentration (ng/μL) in a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). The quality of the DNA yielded by each method was determined by gel electrophoresis in a 1% agarose gel at 90 V for 15-20 min, stained with Ethidium bromide

To confirm the identity of the fungus, ITS-5 (5`-GGAAGTAAAAGTCGTAACAAGG-3`-) and ITS-4 (5`-CCTCCGCTTATTGATATGC-3’) were used to amplify complete internal transcribed spacer (ITS) as described by White et al. (1990). The ITS region was amplified in a 25 μL reaction using 25 ng DNA, 1 x Dream Taq buffer (1.5 mM MgCl₂), 0.3 μM pimer, 200 μM dNTP, 0.2 U Taq DNA Polymerase. The PCR conditions were as follows: 95°C for 1 min, 35 cycles of 95°C for 15 s, 55°C for 30 s, and 72°C for 90 s, and a final cycle at 72°C for 5 min. PCR products were electrophoresed on a 1% (w/v) agarose gel in 1x THE (20 mM Tris-HEPES, pH 8.06) at 90 V for 1 hour. The product was purified and then sequenced. The nitrogen base sequence was analyzed using an automated DNA sequencer. Sequencing data were trimmed and assembled using ChromasPro program version 1.5. The program Molecular Evolutionary Genetic Analysis software, ver. 5.0 (MEGA4.0; http://www.megasoftware.net) was performed in order to edit and align the sequence files, which were manually adjusted. In order to assess the relationships between the major taxa, ambiguous parts of the ITS regions were removed from further analysis and more conserved and alignable parts of the region and all genes were used to generate phylogenetic trees containing representative taxa from major groups. The assembled data were BLASTED with genomic data that has been registered in NCBI/National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/). The aligned sequences were BLAST in GenBank database, to identify all the strains. In this study, phylogenetic tree was generated using maximum parsimony (MP) in MEGA5.0 (Tamura et al., 2011). Bootstrap values for the maximum parsimony tree (MPT) were calculated for 1,000 replicates (Felsenstein, 1985). The edited ITS sequences were compared with other available Fusarium species sequences in the GenBank. Furthermore, the sequences of some known species were downloaded from GenBank and used to reconstruct a combined ITS region and phylogenetic trees.

Sporulation was obtained by culturing the fungal strains in a MEA medium after 7 days of incubation at 28°C (Pujol et al., 1997). Afterwards, the spores were collected by flooding the plate with 1 mL water, including 0.05% Tween 20, using a sterile spread rod. Then, the number of spores was counted manually with a haemocytometer (Merck, Germany) with light microscopy (Optika, Italy) before being diluted to an inoculum of about 10⁶ spores/mL (Noman et al., 2016).

Ryegrass seeds of the English Ryegrass variety were kindly offered by the technical service of the Golf Royal Dar Es-salaam, Rabat, Morocco.

The substrate used for the cultivation of the ryegrass consists of 100% vegetable soil. Topsoil sieved through a 2-mm sieve was autoclaved twice at 121°C/0.1MPa for 30 minutes.

Thirty seeds were disinfected with 0.5% sodium hypochlorite solution for 20 min and then rinsed twice with sterile distilled water for 10 min and dried before being transferred to Petri plates containing sterile layers of filter paper (Kordali et al., 2007; Kordali et al., 2008). Afterward, each group received 1 mL of sterile water daily. After seeds germination, 1 mL of 99% carvacrol diluted in agar 0.2% (1:10) was dispensed into the plates. Afterwards, the infestation of the groups was carried out by adding 1 mL of spores’ suspension (10⁶ spores/mL) of the F. oxysporum, F. solani, and F. nivale and with a consortium (1:1:1) of all the spores’ strains. The experiment was carried out for 4 weeks under ambient temperature (28 ± 2°C). The experimental protocol has been presented in the Table 1 . Plant height (PH), root length (RL), percentage of second leaf appearance (SL) and percentage of second root appearance (SR) were measured weekly. Moreover, the lesions developed on the coleoptile were also noted and the incidence of the disease (I) was estimated by the following equation (Mironenko, 1960):

Where n1 and n2, respectively, represent the total number of seeds per Petri plate and the number of seeds with unaltered coleoptile of seedlings.

One gram of ryegrass’ seeds (approximately 200 seeds) was seeded into pots containing 150 g of topsoil. Each group received a constant volume of 15 mL (in order to wet the soil completely) of carvacrol (diluted in agar 0.2% (1:10)) twice a week. After that, infection was carried out with 15 mL of the spores’ suspension (10⁶ spores/mL) of F. oxysporum, F. solani, and F. nivale and with a mixture of all strain’s spores. The experimental protocol has been presented in the Table 2 . The evolution of the fungal charge of Fusarium was followed once a week using the serial dilution technique. One gram from each sample was transferred to test tube containing 9 mL of 0.9% NaCl. From this solution, a series of dilutions (10^-2 to 10^-6) were made (El Barnossi et al., 2019). From each dilution 0.1 mL was spread on Petri plates containing MEA medium. The plates were incubated at 27°C for 4 days. Colonies of Fusarium species were then counted by the enumeration technique. Consequently, plates containing 15 to 150 colonies were selected for counting and the result was expressed in log (CFU/g) of three replicates (Ribeiro et al., 2020). As regards the virulence of each fungal strain, it was estimated after 7 weeks based on the disease index (DI) technique using the 0-4 scale established by Hernandez et al. (1998) which was based on visible symptoms on each seedling, where code 4: dead turf, code 3: 67 to 99% affected plant, code 2: 34 to 66% affected plant, code 1: 1 to 33% affected plant and code 0: no visible symptoms. The experiment was carried out under greenhouse conditions using both normal topsoil and disinfected topsoil.

To determine the fresh shoots and roots weight, the shoots and roots were carefully washed under running tap water. Prior to weight determination, excess moisture was blotted using paper towels, and the weight was determined. While for the dry weight determination, the samples were dried in an oven at 70°C for 72 h till constant weight was reached after the dry weight was determined (Huang et al., 2017).

Quantitative results were expressed as means, of triplicate experiments ± SD (standard deviation). To determine statistical significance, Two way-ANOVA and Tukey’s multiple range tests at p< 0.05 were performed using GraphPad Prism 8.0.1 (Graph Pad Software Inc., San Diego, USA).

Molecular identification was confirmed by sequencing the ITS. BLAST analysis using ITS sequences revealed a threshold of 100% similarity between the sequences from the isolates in this study and sequences from strains which were previously deposited in Genbank. The ITS sequences were deposited in GenBank as: F. solani (accession no. OP810954), F. oxysporum (accession no. OP824842), and F. nivale (accession no. OP810953).

All strains isolated from soils and ryegrass represented two different lineages ( Figure 1 ). The isolates of F. solani and F. oxysporum were divided into subgroups. F. nivale was included in other branches. Phylogenetic tree demonstrated that all strains are closely related with a strong bootstrap support (100%). All isolates described as F. solani, F. oxysporum and F. nivale, based on morphological characteristics, were identified as F. solani, F. oxysporum and F. nivale through molecular analyses.

Table 3 shows the results of the effect of carvacrol treatment on the coleoptile growth and on rhizogenesis of ryegrass in-vitro. After infestation of the seedlings with fungal spores, Fusarium significantly reduced plant height and root length for all tested strains compared to the negative control, especially F. nivale which was the most virulent on ryegrass seedlings. In contrast, after treatment, plant height and root length recovered well with all tested concentration of carvacrol. As a consequence, the measurement values of the treated plants were almost equal to those of the negative control and sometimes better.

On the other hand, Table 3 shows also the results of the carvacrol effect on plant development. According to the result portrayed by the table plants in both the positive and negative control did not develop a second leaf. However, in the treated groups between 10 and 23.33% of the plants have developed a second leaf during the experiment time. In addition, as regards the carvacrol effect on the appearance of the second root, 10% of the plants in the negative control developed the second root. After infestation with F. nivale, the fungus inhibited the development of the second root, and consequently the percentage was decreased to 1.37%. Treatment with 0.05, 0.1, and 0.2 mg/mL carvacrol significantly allowed the emergence of the second root in all plants with a percentage reaching 20.33, 26.67 and 34% respectively.

Regarding the incidence of lesions on coleoptiles of ryegrass seedlings, the Figure 2 shows that the incidence was high with F. nivale with a percentage of 50%. Fusarium solani, F. oxysporum and the spores mixtures did not induce severe lesions on coleoptiles, where the incidence percentage ranged between 23 an 33%.

According to the results, carvacrol showed protective effect on ryegrass against Fusarium wilt disease. Furthermore, carvacrol showed growth-promoting effect on both the leaves and the rhizogenesis.

To evaluate the preventive effect of carvacrol and the virulence of the Fusarium species, the disease index (DI) was used. This index is an indication of the health status of the plant, the more it tends towards 4 the more affected the plant is. The results presented in Figure 3 demonstrate that before infestation, the DI was equal to 2, whereas, after the infestation the DI was increased to 3 and 4. In contrast, after the treatment with carvacrol, the DI decreased to 1 and 0. In the disinfected soil, F. nivale - with a DI tending towards 4 - was found to be the most virulent, however in the normal soil this virulence was reduced which was reflected by the elevation of DI to 2 as the negative control. The aggresivity of F. nivale is manifested by the total death of almost all plants of the infested and untreated lot in the disinfected soil ( Figure 4 ). Regarding the most effective concentrations of carvacrol, 0.1 and 0.2 mg/mL were shown to have a better protective effect with a DI ranging between 1 and 0 against all tested strains and in both normal and disinfected soil.

Figures 5 , 6 show the appearance of the health status of all pots in the greenhouse in disinfected and normal soil. The pictures show that the plants in the infested and treated lots with carvacrol were healthier than those in the infested and untreated lots.

The fungal load of Fusarium was measured during the whole period of ryegrass cultivation. Figures 7 , 8 show the results of the follow-up of this load in both disinfected and normal soil. According to the figures, after the infestation, the Fusarium load was much higher in the disinfected soil than in the normal soil compared with negative control. In the normal soil, after treatment, the load of F. solani became null from the third week of treatment with 0.2 mg/mL and at the end of the fourth week the load was nulled with 0.1 mg/mL of carvacrol. In the disinfected soil, F. solani was eliminated with 0.2 mg/mL of carvacrol from the second week. Furthermore, with this concentration, the fungal load of all the spores’ mixture was well eliminated after three weeks in both soil types. We noticed also that the disinfected soil remained without any apparition of fungal charge until the last week, we found a little of Fusarium charge ( Figure 8 ).

Figures 9 , 10 show the results of the quantitative evaluation of the vegetative growth variables such as fresh and dry weight of ryegrass shoots and roots after 4 weeks of treatment with carvacrol in normal and disinfected soil under greenhouse. In Figure 9 , where we used normal soil, after infestation with Fusarium spores, the amount of organic matter has reduced significantly for all the groups and also for all growth parameters. After treatment, the amount of organic matter recovered and sometimes even increased especially with 0.1 and 0.2 mg/mL of carvacrol, compared to the negative control. On the other hand, in disinfected soil ( Figure 10 ), F. nivale showed more damage on ryegrass. As a result, the amount of organic matter decreased from 7.4 to 4.9 g, from 2.17 to 0.26 g and from 1.87 to 0.17 g for the fresh shoots weight, dry shoots weight and dry root weight respectively.