*2.4. Assessment of Antagonistic Effectiveness of CARA17 Strain on Foeniculum vulgare L. Seedlings against Fungal Soil-Borne Pathogens In Vivo*

The results obtained from in vivo experiments carried out on fennel seedlings are reported in Table 2. The symptoms observed on the root consisted of browning, rot, and growth reduction, while on the leaves they consisted of general growth reduction and yellowing of the epigeal portion (Figure 2). The DS values collected by observations carried out on the root of seedlings treated by dipping in CARA17 inoculum suspension were 0.0 for *A. rolfsii* and *S. sclerotiorum*, from 0.6 to 0.9 for *P. ramiseptata*, *V. dahliae* and *F. oxysporum*. Disease severity values observed on roots increased for experiments where the CARA17 strain was added to a pot after the transplanting of fennel seedlings. In particular, DS values varied from 0.6 to 1.9 against *S. sclerotiorum*, *V. dahliae*, *F. oxysporum* and *P. ramiseptata*. No DS value was produced by *A. rolfsii.* The DS values were higher when no CARA17 inoculum was used against the fungal soil-borne pathogens. The most aggressive fungal pathogen was *A. rolfsii* with a DS value of 4.8, and the less aggressive *S. sclerotiorum* with a DS value of 2.1. No DS values were recorded from fennel seedlings used as control not treated. The DS values recorded from leaves showed the same trend observed on the root, although they resulted in being lower than in those collected from roots (Table 2). The percentages of re-isolation of fungal soil-borne pathogens were significantly higher when the plants were not treated with both kinds CARA17 inoculations (100.00% for *A. rolfsii*; 80.00% for *V. dahliae*), while they were lower when the seedlings were treated with CARA17 by dipping inoculation (13.3% for all fungal pathogens except for *V. dahliae*), and were major with CARA17 inoculated by pouring into pots after transplanting (26.6% for *A. rolfsii*, and 80.00% for *V. dahliae*) (Table 2).

**Figure 2.** Effectiveness of CARA17 strain as antagonist for controlling disease severity by dipping treatment and after transplanting of fennel seedlings. Fennel seedlings not treated (**a**), treated with CARA17 strain by dipping (**b**) and after transplanting (**c**). Fennel seedlings treated by only fungal pathogen (1); fungal pathogen with CARA17 strain by dipping (2) and after transplanting (3). Inoculation with: *Athelia rolfsii* (**d1**–**d3**); *Fusarium oxysporum* (**e1**–**e3**); *Plectosphaerella ramiseptata* (**f1**–**f3**); *Sclerotinia sclerotiorum* (**g1**–**g3**); *Verticillium dahliae* (**h1**–**h3**).


 on fennel

**Table 2.**

Effectiveness

 of CARA17 strain as antagonist for controlling

 disease severity (DS) against fungal soil-borne pathogens

#### *2.5. Plant Growth Promotion by Streptomyces Strain In Vivo*

Table 3 also reports the data related to the promotion of plant growth by the evaluation of fresh and dry biomasses (related to the entire plants). According to Shapiro–Wilk tests, the data from fresh and dry biomasses followed normal distributions (fresh biomass: W = 0.90, *p* < 0.01; dry biomass: W = 0.88; *p* < 0.01). The Levene tests showed that the homogeneity of variances, from the in vivo antagonism experiments (TEST1 and TEST2) carried out on fennel seedlings, were significant (fresh biomass: F = 3.74, *p* < 0.01; dry biomass: F = 3.75, *p* < 0.01). One-way ANOVA highlighted the inoculation by the CARA17 strain significantly promoting the growth of fennel seedlings also in the presence of fungal soil-borne pathogens.

**Table 3.** Effectiveness of CARA17 strain on promotion of plant growth by biomass production of fennel seedlings.


\* Biomass includes epigeal and hypogeal plant tissues; \*\* mean values of 15 replicates; § Standard Deviation; † Data followed by different capital letters within the column are significantly different (Fisher's tests; *<sup>p</sup>* < 0.01).

The lowest biomasses produced (fresh, 38.13 g; dry, 4.15 g) were recorded from seedlings treated only with *A. rolfsii* with respect to other fungal soil-borne pathogens used alone without any antagonistic treatment (Figure 2). Indeed, the fresh/dried biomasses varied from 44.08/4.89 to 48.33/6.03 g, when the seedlings were treated with the other fungal soil-borne pathogens. In general, the biomasses resulted in being higher when the fennel seedlings were subjected to dipping in CARA17 inoculum rather than to being poured into pots after transplanting. In particular, the trials where the CARA17 strain was used as the dipping inoculation of seedlings inoculated with *A. rolfsii* as fungal soilborne pathogen, allowed obtaining the highest biomasses (fresh, 93.11 g; dry, 11.69 g). The biomass values (fresh/dry) from other trials treated with the dipping of CARA17 inoculum and other fungal pathogens varied from 85.07/6.90 g (*F. oxysporum*) to 87.27/8.23 g (*S. sclerotiorum*).

The treatment of the CARA17 strain by dipping without fungal pathogens allowed the fennel seedlings to reach significantly higher biomass values (fresh/dry, 165.03/14.43 g) than during treatment after transplanting (fresh/dry, 122.86/12.74 g), and no antagonistic treatment (fresh/dry, 101.61/11.66 g) (Table 3).

#### **3. Discussion**

Since in agriculture, low environmental impact control means that phytopathogenic agents are increasingly in demand to reduce the negative effects of chemicals such as pesticide residues in plant products for human use and to preserve the natural ecosystems, the present work reports preliminary results obtained from biological assays carried out in in vivo and in vitro conditions. It is known that biological control by microorganisms such as *Streptomyces* spp. confirms that they are a promising tool for the management of various microbial diseases causing severe losses of agricultural yields. For this reason, the development of biological control (BCAs) and plant growth promoting agents are needed [19]. To date, a very large number of *Streptomyces* strains as antagonists have been used to promote plant growth and control soil-borne phytopathogens [20]. In the present work, a *Streptomyces* strain named CARA17 isolated from some healthy roots of grapevine affected by grapevine trunk diseases was subjected to identification and then used to ascertain its capacities to control five soil-borne phytopathogens, known to be ubiquitous and polyphagous. With cultural and molecular tools, the strain CARA17 was found to belong to the *Streptomyces albidoflavus* group, which was used to ascertain if it was able to control *A. rolfsii*, *F. oxysporum*, *P. ramiseptata*, *S. sclerotiourum* and *V. dahliae* as severe fungal phytopathogens. Antagonism assays carried out in dual cultures in in vitro conditions showed that the *Streptomyces* CARA17 strain was capable of inhibiting the mycelial growth of soil-borne pathogens used as targets with varying degrees of inhibition. As it was observed that the CARA17 strain could inhibit the mycelial growth after some days, in order to emphasize its antagonistic capability, it was placed in Petri dishes 14, 21 and 28 days before the placing of mycelia plugs of phytopathogens. Indeed, it was possible to assess that the CARA17 strain showed a high ability to control the fungal mycelium starting from 14 days after its placement in a Petri dish. It is probable that the CARA17 strain releases bioactive antimicrobic compounds in an artificial medium some days after its placement according to Kaur et al. [21], who found that *Streptomyces* spp. are able to produce a large number of metabolic compounds. In particular, these researchers assessed the antifungal activity of *Streptomyces* spp. against *Fusarium moniliforme* which is responsible for Fusarium wilt in tomato plants in both in vitro and in vivo experiments. Moreover, other investigations reported the effectiveness of *Streptomyces* strains for managing rice blast disease caused by the fungus *Magnaporthe oryzae* [22] in greenhouse conditions, and considering these actinobacteria excellent candidates as biocontrol agents. According to the latest research, our findings allowed us to observe that the CARA17 strain was able to inhibit *S. sclerotiorum* all three times (14, 21 and 28 days of exposition) while against the other fungal pathogens the antagonistic effectiveness was weakly reduced but still significant, and decreased weakly from 14 days to 28 days of exposition. In dual culture, only *A. rolfsii* was already poorly controlled at 14 days of exposition, and not inhibited at 21 and 28 days.

In in vivo conditions, the CARA17 strain was also demonstrated to be efficacious at controlling all fungal phytopathogens used in the present work, although the best effectiveness consisted of the control of *Scleotinia sclerotiorum* for both trials—the dual culture and the greenhouse experiment. Lower but significant biocontrol action acted on by the CARA17 strain was detected against *F. oxysporum*, *P. ramiseptata* and *V. dahliae*. Similar results have been discussed by Colombo et al. [15] when they used two *Streptomyces* strains to control *Fusarium graminearum* as an agent of FRR (Fusarium root rot), FFR (Fusarium foot rot) and FHB (Fusarium head blight) in greenhouse and open field conditions. While no protection was assessed against *A. rolfsii*, a considerable effect of growth promotion in fennel seedlings was observed. The biological control played by the CARA17 strain on fennel seedlings artificially inoculated with fungal soil-borne pathogens was more clear in terms of the protection of seedlings by dipping inoculation before transplanting than by pouring the inoculation into pots after transplanting. The CARA17 strain was probably more able to protect the root of fennel seedlings by dipping inoculation, because the antifungal compounds present in the inoculum solution were immediately adsorbed by roots enhancing the hyperparasitism mechanism and inducing systemic resistance [23,24].

Further, the greenhouse experiments determined significant effects of *Streptomyces albidoflavus* CARA17 strain on the growth parameters of the fennel seedlings, such as the hypogeal and epigeal portions and the fresh and dry weights of the biomasses. Therefore, the data obtained from the in vivo experiments suggested that the CARA17 strain might be used as a biological control agent (BCA) and plant growth promoting agent (PGPA) when used as a propagules inoculum containing spores or mycelium. It is known that *Streptomyces* spp. are included in the PGPR microbial community (Plant Growth Promoting Rhizobacteria), and they are actively or passively involved in plant growth promotion [25]. Therefore, in this work, by the fresh and dry biomass weights obtained, it was possible to assess the ability of the CARA17 strain: (a) to promote the fennel seedlings growth, because it may act as a biofertilizer; (b) to facilitate the tolerance to biotic (phytopathogens used in vivo conditions) and abiotic stresses (no organic and mineral fertilizers), because the disease severity indices collected from the fennel seedlings were significantly reduced due to the presence of the CARA17 strain.

Moreover, this study allowed us, for the first time, to associate *Plectosphaerella ramiseptata*, as a soil-borne pathogen, with fennel plants, and to ascertain its ability to cause significant damages to seedlings consisting of root browning, leaf yellowing and plant growth reduction. Similar symptoms and the disease severity caused by *P. ramiseptata* have also been reported in tomato, pepper, basil and parsley [26,27].

The preliminary results discussed here encourage further studies to assess whether the CARA17 strain is able to produce putative antifungal compounds, to extract secondary metabolites as putative resistance inducers, and to verify if biocontrol and growth promotion actions can be improved and increased by formulation with other PGPR microorganisms.
