4.7.5. Disease Severity Assessment

For treated seedlings inoculated with *M. phaseolina*, disease severity (DS) was assessed at 35 days after inoculation using the following DS rating scale: 1 = no visible disease symptoms; 3 = wilt restricted to cotyledons, lower stem tissues with small necrotic lesions; 5 = 10% of hypocotyl and lower stem tissues showing lesions, fungal fruiting structures starting the development in the affected tissues, 7 = 25% of hypocotyl and lower stem tissues showing lesions, with development of fungal fruiting structures in the affected tissues; and 9 = ≥50% of hypocotyl and lower stem tissues with lesions, with abundant development of fungal fruiting structures [57]. Subsequently, a DS index was estimated using the following formula:

$$\text{DS}(\%) = \left[ \sum\_{i=1}^{9} n\_i(st\_i) / (N \times K) \right] \times 100$$

in which *ni* = number of seedlings in the DS development stage *i*, *sti* = value of the DS stage (1–9), *N* = total number of plants assessed, and *K* = largest scale level (9) [58].

Regarding treated seedlings inoculated with *R. solani*, DS was evaluated separately on stem and roots tissues at 28 days after inoculation by using the following DS rating scales: (i) DSstem: 1 = absence of lesions on hypocotyl, 2 = superficial lesions (yellow-brown discoloration) on hypocotyl, 3 = deep tissue lesions, and 4 = seedlings dead or wilted [59]; (ii) DSroot: 0 = healthy seedlings, 1 = yellowish-brown discoloration near hypocotyl, 2 = yellowish-brown discoloration plus lesions or brown spots near hypocotyl, 3 = entirely brown surface or lesions covering more than 75% of root surface, and 4 = pre-emergence damping off, seedlings dead or wilted [60]. Subsequently, a DS index was estimated for each tissue using the following formulas:

$$\text{DSstem}(\%) = \left[\sum\_{i=1}^{4} n\_i(st\_i) / (N \times \text{Kstem})\right] \times 100$$

$$\text{DSroot}(\%) = \left[\sum\_{i=1}^{5} n\_i(st\_i) / (N \times \text{Kroot})\right] \times 100$$

in which *ni* = number of stems or roots in the DS development stage *i*, *sti* = value of the DS stage (1–4 and 0–4 for stems and roots, respectively), *N* = total number of plants assessed, and *K* = largest scale level (4 in all cases) [58]. Furthermore, for each combination of soil and treatment, the incidence of disease (DI; % of affected plants) and mortality (% of dead plants) were estimated at 28 days after inoculation.

Ungerminated seeds or plants with lesions on the hypocotyl, roots and/or stem were subjected to wet chamber and microscope preparations to confirm the identity of the inoculated pathogens.

#### *4.8. Data Analyses*

Data from the repetitions of each experiment were combined after checking for homogeneity of the experimental error variances by the *F* test (*p* ≥ 0.05). Subsequently, data were tested for normality and homogeneity of variances prior to conduct analyses of variance (ANOVA). For the dual culture assay, factorial ANOVA was conducted with MGI as dependent variable, and actinobacterial strains, fungal pathogens and their interaction as independent variables. Significant differences were observed for the two independent variables as well as for their interaction (*p* < 0.0001 in any cases). Thus, independent ANOVA were conducted to determine differences between actinobacterial strains against each fungal pathogen. For each fungal pathogen, mean values were compared using Tukey's honestly significant difference (HSD) tests at *p* = 0.05 [61]. For the enzymatic activity, data of the halo (mm) for each of the three parameters evaluated were analyzed separately by the non-parametric Kruskal-Wallys test due to the assumptions of normality and homogeneity of variances were not fulfilled even though logarithmically, arcsine or square root transformation of the data were conducted. Data from the actinobacterial strains that not develop halo (0.0 mm) were excluded from the statistical analysis in any cases. Mean values were compared using Dunn's comparisons test at *p* = 0.05. In the *in planta* experiment, data of total DS (seedlings inoculated with *M. phaseolina*), and DSstem and DSroot (seedlings inoculated with *R. solani*) were tested for normality and homogeneity of variances prior to conduct analyses of variance (ANOVA). Data from negative control were omitted since no symptoms were observed in all cases. For each dependent variable, a split-plot ANOVA was conducted with soil (*n* = 2) as main-plot factor and treatments (*n* = 6) as the subplot factor. Due to significant differences were observed in all cases for the two independent variables as well as for their interaction (*p* < 0.005), independent ANOVA were conducted to determine differences between treatments for each disease. The treatment means of total DS, or DSstem and DSroot were compared according to Fisher's protected LSD test at *p* = 0.05 [61]. For both inoculated plants with *M. phaseolina* and *R. solani*, data on the final DI (% of affected plants) and mortality (% of dead plants) were analyzed by multiple comparisons for proportions tests at *p* = 0.05 [62]. Additionally, for plants inoculated with *R. solani*, the Pearson correlation coefficients (*r*) between the DSstem

and DSroot were calculated using the average values of the two variables for each of the treatment evaluated in sterilized or non-sterilized soil (*n* = 6 in each type of soil). All data analyses were conducted using Statistix 10 [63].
