*4.5. In Vivo Growth Promoting Effect and Disease Control*

An in vivo pot experiment was carried out in a greenhouse (School of Agricultural, Forestry, Food and Environmental Sciences-SAFE, University of Basilicata, Potenza, Italy) to evaluate the growth-promoting effect (GPE) of the tested *Actinomycetes* isolates on tomato plants, and the disease control (DC) of the most bioactive isolates was studied against *S. sclerotiorum*.

The pot experiment was carried out in a glass greenhouse at 25 ◦C for a 15-h photoperiod. Each pot was 20 cm high and 25 cm wide, and previously sterilized with 1.2% sodium hypochlorite for 5 min, rinsed twice with distilled water, and filled with a growing medium mixture (compost/peat moss, 1:1). Seeds of *Solanum lycopersicum* L. cv. *cerasiforme* were surface sterilized by ethanol (70%) and sowed in a cell tray. The temperature and relative humidity in the greenhouse remained stable at 25 ± 2 ◦C and 70–80%, respectively, for the duration of the experiment.

For the *Actinomycetes* treatment, an initial nutrient culture of peptone yeast calcium agar (PY-Ca) was prepared for the tested isolates and incubated for 5 days at 28 ± 2 ◦C. A suspension of each studied isolate was prepared by inoculating 10<sup>6</sup> CFU/mL from the original culture into minimal mineral (MM) media prepared as follows: (g/L) 10.5 K2HPO4, 4.5 KH2PO4, 1.0 (NH4)2SO4, 0.5 Na3C6H5O7 × 2H2O, 0.2 MgSO4 and 5.0 dextrose. The pH value was adjusted at 7.0. The suspensions were then incubated for 7 days at 28 ± 2 ◦C. The broth cultures were poured into the rhizosphere zone of tomato seedlings (100 mL/pot) 15 days after germination (DAG).

For the fungal artificial infection, Ø 5 mm agar discs from a pure fresh culture (96 h) of *S. sclerotiorum* were inoculated in a sterilized flask filled with potato dextrose broth (PDB) and incubated for 7 days at 22 ± 2 ◦C. After that, 50 mL of the incubated broth was inoculated in the rhizosphere zone of the seedlings 10 days after the *Actinomycetes* treatment. Ten seedlings were used as the negative health control. The whole experiment was repeated twice with five replicates per treatment. The experimental pots were distributed in a randomized block design in the greenhouse and watered once a day.

For the eco-physiological parameters, plant growth was monitored at the end of the experiment, about 40 DAG, by measuring stem length (SL) in centimeters, number of leaves (NL), number of twigs (NT), the total fresh weights of shoots (TFwS) in grams, and total dry weight of shoots (TDwS) in grams. Regarding the evaluation of the disease incidence, tomato plants were monitored daily, fifteen days after the infection (DAI), to observe the eventual appearance of disease symptoms. The disease incidence was assessed using the following scale (0 = no symptoms observed; 1 = 1 to 20% of leaf chlorosis; 2 = 21 to 50% of leaf chlorosis; 3 = 51 to 80% of leaf chlorosis; 4 ≥ 80% of leaf chlorosis), as reported by Elshafie et al. [4]. The infection percentage (IP %) was measured using Equation (1), whereas the disease index (DI %) and the control effect (CE %) were calculated using Equations (2) and (3), respectively, as described by Lee et al. [52].

$$\text{IP}\,\%=\text{(SL/TL)}\times100\tag{1}$$

$$\text{IDI\%} = \left[ \sum \text{(Scale} \times \text{No. of SL)} / \text{(HS} \times \text{TL)} \right] \times 100 \tag{2}$$

$$\text{CE } \%= 100 \times \text{(DI.P}-\text{DI.B)}/\text{DI.P} \tag{3}$$

where SL is symptomatic leaves; TL is total number of leaves; HS is highest scale; DI-P is disease index of infection; DI-B is disease index of control.
