Rose bengal chloramphenicol agar

Plate Count Agar (PCA, Oxoid Limited, Hampshire, UK) was used for the enumeration of total aerobic mesophilic bacteria and the plates were incubated aerobically at 37°C for 48 h. For total psychrotrophic bacteria, PCA (Oxoid, UK) was used as well and the count was enumerated at 7°C for 10 days. Pseudomonas were determined on CFC Agar (Pseudomonas Agar Base CM 0559-Oxoid) with Selective Agar Supplement (SR0103, Oxoid, UK) and the plates were incubated under aerobic conditions at 25°C for 48 h. Violet Red Bile Dextrose Agar (VRBD Agar, Merck KGaA, Darmstadt, Germany) was used for Enterobacteriaceae and the plates were incubated 48 h at 30°C under anaerobic conditions (Anaerocult A, Merck, Germany). Also, for lactic acid bacteria, Man Rogosa Sharpe Agar was used (MRS, Oxoid, UK) at 30°C for 48 h under anaerobic conditions. Rose-Bengal Chloramphenicol Agar (RBC, Merck, Germany) was used to determine the count of yeast/mold and plates were incubated at 25°C for 5 days under aerobic conditions.

Cultures of Fusarium oxysporum grown, for 7 days at 25 °C in Potato Dextrose Agar (PDA, Merck, KGaA, Darmstadt, Germany ), were used to prepare the conidia suspension as described in the literature [36 (link)]. The absence of hyphae in the suspensions was checked via light microscopy (Leitz Laborlux K, Ernst Leitz GmbH, Wetzlar, Germany). The concentration of the viable conidia was determined with the serial dilutions of an aliquot in phosphate saline buffer (PBS, pH 7.4) and spread-plated on Rose Bengal Chloramphenicol Agar (Merck, KGaA, Darmstadt, Germany). Colonies were counted after 2 days of incubation at 25 °C, and the concentration of conidia is expressed as colony forming units per milliliter (CFU·mL⁻¹) of suspension.

Ten-fold dilutions of soil suspensions were prepared in sterilized tap water. One hundred microliters of each dilution was used for the inoculation. Three plates were used for each dilution. To assess the CFU of spore-forming bacteria, the dilution series were heat treated at 90°C for 10 mins before spreading onto the medium. The number of heterotrophic bacteria and spore-forming bacteria (CFU) were measured on nutrient agar (Merck Millipore, Germany), and that of fungi on Rose Bengal Chloramphenicol agar (Merck Millipore) after a 5-d inoculation period at 25°C.

Substrate samples were collected from the plant rhizosphere at the first floral sampling and the end of cultivation. Fungus quantification was performed by serial substrate dilutions in a Trichoderma-selective agar medium according to Fiorentino et al. [59 (link)], with some modifications. In detail, 10 g of root–substrate was suspended in sterile distilled water to provide serial dilutions (four replicates per dilution), and 100 μL aliquots of each sample were spread on the surface of 90 mm culture plates containing Rose Bengal Chloramphenicol agar (Merck KGaA, Darmstadt, Germany). The plates were incubated at 25 °C and examined daily for emerging fungal colonies. The results have been expressed as the CFU per g of dry substrate.

Measurement of moisture and ash was performed according to AOAC [55 ] and Ho et al. [56 ]. Additionally, to determine the mold count, 1 mL of the sample was pour-plated in Rose-Bengal chloramphenicol agar (Merck, Darmstadt, Germany) and then incubated at 22 ± 1 °C for 5–6 days. Finally, mold colonies were counted [57 (link)].