*4.5. Assessment of Antagonistic Effectiveness of CARA17 Strain on Foeniculum vulgare L. Seedlings against Fungal soil-borne pathogens In Vivo*

The inoculum solution with the CARA17 strain propagules was prepared by collecting spores and small fragments of mycelium scraped from surface of 21-day-old *Streptomyces* colonies grown on PDA medium at 28 ± 3 ◦C in darkness until reaching a concentration of <sup>1</sup> × 107 cfu/mL in sterile Tween 20 solution (0.2%).

*Inoculation preparation of fungal soil-borne pathogens.* The inoculum solution with each fungal soil-borne pathogen was prepared as described above for the CARA17 strain, scraping from the surface of 21-day-old colonies grown on PDA medium at 21 ± 3 ◦C in darkness until reaching a concentration of 1 × 107 cfu/mL in sterile Tween 20 solution (0.2%).

The experimental design was performed as two independent batches at the end of August and consisted of two different inoculation kinds, where the horticultural host target was represented by 30-day-old seedlings of *Foeniculum vulgare* var. DONATELLO F1 (HM.CLAUSE Vegetable Seeds).

The first experiment (TEST1) consisted of preliminarily dipping the fennel seedlings in the inoculum solution of the CARA17 strain for 30 min, before transplanting them in a pot containing 1.5 kg of soil and peat (3:1), (sterilized early twice at 121 ◦C for 30 min and kept for 20 days in a controlled chamber at 25 ± 3 ◦C, 70% relative humidity, and under natural light), and wetting them with 500 mL of irrigation water. Subsequently, 50 mL of inoculum solution of each fungal soil-borne pathogen was poured into the soil of each pot around the collar of the fennel seedlings.

The second experiment (TEST2) consisted of inoculation with 50 mL of inoculum solution of each fungal soil-borne pathogen after seedlings transplantation into wet soil with 500 mL of irrigation water. After 48 h, 50 mL of CARA17 inoculum solution was poured into the soil around the collar of the fennel seedlings.

As control trials, for both experiments (TEST1 and TEST2), pots containing fennel seedlings treated with the CARA17 strain, treated with each fungal pathogen, and not treated with either the CARA17 strain or the fungal soil-borne pathogens, were prepared. Each trial was replicated fifteen times. The pots with fennel seedlings were placed in a greenhouse with temperature and humidity not conditioned. During the growth of the seedlings, no fertilizers, pesticides or fungicides were used. They were only subjected to irrigation practice when necessary, using the same water volumes for each pot. After 100 days, the fennel plants were gently removed from the pots, the roots and collars were carefully washed, and the presence/absence of browning symptoms observed on the root and collar were evaluated and described using an empiric scale from 0 to 5, where 0 = no symptoms observed; 1 = 1–20%; 2 = 21–40%; 3 = 41–60%; 4 = 61–80%; and

5 = 81–100%. The disease severities (DS) on the roots and collar were determined according to the following formula:

$$\text{DS} = \frac{\sum (Number\ of\ observed \times values\ of\ scores)}{\text{Total number of cases}}.\tag{1}$$

All fungi underwent re-isolation from the root, collar and stem of the inoculated plants to fulfil Koch's postulates.
