*4.2. In Vitro Effect of Actinobacterial Strains against Macrophomina phaseolina and Rhizoctonia solani: Dual Culture Assays*

All the 60 actinobacterial strains (Table 6) were evaluated for their effectiveness inhibiting mycelial growth of *M. phaseolina* isolate CCIBP-Mp1 and *R. solani* isolate CCIBP-Rh1 by means in vitro dual culture assays. The two pathogenic fungi were obtained from the collection of plant pathogenic fungi of the Instituto de Biotecnología de las Plantas (IBP) of the Universidad Central Marta Abreu de Las Villas (Cuba), where are maintained growing on PDA at 5 ◦C in darkness. These isolates were selected due to their high aggressiveness previously tested in the common bean crop [36].

Prior to conduct the dual culture assay, the 60 actinobacterial strains were grown on CSA (pH = 7) at 30 ◦C for seven days in darkness. The inoculum of *M. phaseolina* and *R. solani* was prepared by seeding suspensions of mycelial fragments of each isolate on Potato Dextrose Agar (PDA; BioCen, Bejucal, Mayabeque, Cuba) at 28 ◦C for three days in darkness. In vitro dual culture assays were conducted in 9.0 cm in diameter Petri dishes with PDA [37]. To this end, a 7.0 mm in diameter mycelial plug of the pathogen was placed at one end of the plate, and another 7.0 mm in diameter mycelial plug of the actinobacterial strain was plated at 50.0 mm apart at the opposite end. Additionally, 7.0 mm in diameter mycelial plugs of *M. phaseolina* or *R. solani* isolates were seeded in the center of PDA plates without actinobacteria as a positive growth control. All Petri dishes were incubated at 28 ◦C in total darkness, and the radial mycelial growth of the two plant pathogens was assessed every 24 h, until seven days of incubation [38,39]. There were three replicated Petri dishes per actinobacterial strain (*n* = 60) and plant pathogen (*n* = 2) or control (*n* = 2) combination in a completely randomized design [(60 actinobacterial strains × 2 fungal pathogens × 3 Petri dishes) + (2 control × 3 Petri dishes) = 366 Petri dishes in total]. The experiment was performed three times under similar conditions.

For each fungal pathogen, the percentage of the inhibition of mycelial growth was calculated using the following formula:

Mycelial growth inhibition (MGI) (%) = [(RGR-rgr)/RGR] × 100

where 'rgr' is the radial growth of *M. phaseolina* or *R. solani* in dual culture with each actinobacterial strain, and 'RGR' is the radial growth rate of the control treatment (fungal pathogen isolates growing on PDA without actinobacterial strains).
