The strain V26, used throughout this study, was isolated from the rhizosphere of untreated almond tree (Bir El Malouli, Sfax, Tunisia) and identified by the laboratory of Biopesticides as B. subtilis [21]. The isolate of R. solani used in this study was obtained from potato, cv. Spunta, a tuber showing typical black scurf symptoms, from Essaïda, a Tunisian potato-growing area. The fungal pathogenicity was previously confirmed on potato plants by Daami-Remadi et al. [9]. The R. solani isolate was grown on Potato Dextrose Agar (PDA) at 25 °C for seven days and stored at 4 °C until use.

A single colony of strain V26, picked from the agar plate, was transferred to a test tube containing 3 mL of LB medium and incubated overnight at 30 °C. The overnight culture was then seeded into a 1000-mL Erlenmeyer flask containing 100 mL of LB medium. The shake-flask cultures were maintained in a shaker set at 200 rpm for 24 h at 30 °C. The overnight cultures were then inoculated into 2-L shake flasks containing 500 mL of LB to achieve the initial cell density of 0.15 at OD_600 nm. The inoculum culture was transferred into a 7-L Labfors (Infors, Switzerland) fully automatically controlled fermenter containing 5 L of culture medium. The production medium consisted of 25 g/L sucrose, 20 g/L peptone, 4.5 g/L yeast extract, 2 g/L KH₂PO₄, 0.6 g/L MgSO₄ and 6 g/L MnSO₄ [22]. The temperature was automatically controlled at 30 °C. The airflow rate and the impeller speed were set at 1 vvm and 200 rpm, respectively [23]. The pH was maintained at 7 with continuous regulation using 2 M HCl and 2 M NaOH. Foam production was controlled by automatic addition of a sterile antifoam solution.

The antifungal activity was assessed in vitro by the well diffusion method [24]. B. subtilis V26 culture medium was centrifuged at 4000 × g for 20 min and the supernatant was recovered. The supernatant was sterilized through a Millipore filter (0.45 μm), then was serially diluted (1:4, 1:8, 1:16, 1:32) with sterile distilled water and used for activity assay. A suspension of R. solani was prepared by suspending loopfuls of hyphae from a Potato Dextrose Agar (PDA) (Lab M) slant. Hundred microliters of R. solani suspension were displayed on a plate filled with the PDA medium amended with chloramphenicol (30 μg/mL). Wells were drilled in the PDA, covered with the R. solani suspension, and then 80 μL of diluted supernatant were added to the wells. The inhibition zones were observed. Each diluted supernatant was assayed in triplicate and the diameter of the inhibition zone was measured after incubation at 25 °C for three days.

The ability of B. subtilis V26 to produce chitosanase was examined on Luria Broth (LB) agar plates: yeast extract 5 g/L, peptone 10 g/L, NaCl 5 g/L, and agar 15 g/L, containing 1.5% (v/v) of colloidal chitosan (Sigma, Deisenhofen, Germany). Protease activity was determined on agar containing 5% (v/v) skimmed milk. Wells were drilled in the PDA plates containing colloidal chitosan or skimmed milk, and then 80 μL of diluted supernatant were added to the wells. An equal volume of culture medium served as a control. The formation of clearing zones was detected after 1 day of incubation in the dark at 30 °C. Each experiment was replicated three times and the assay was repeated twice.

The antagonistic effect of B. subtilis V26 against R. solani was evaluated in vitro by a growth inhibition assay [25]. Increasing volumes of B. subtilis V26 supernatant (1%, 3%, 5%, 7%, 9%; v/v) were mixed with molten PDA (cooled at 45 °C). After cooling, the plates containing the amended PDA were centrally inoculated with a 6-mm agar disc from a fresh culture of R. solani, and were incubated at 25 °C. An equal volume of PDB (Potato Dextrose Broth) was used as a control. Each treatment was done in triplicate and the antagonism assay was repeated twice. The colony's diameter was recorded after 72 h of incubation, in the dark, and the inhibition ratio was calculated using the following formula [26]:

Two 6-mm agar disks of R. solani were transferred into a 250-mL flask containing 50 mL of the PDB medium, and each flask was treated with one of a series of sterile culture supernatants from 1 to 9% (v/v). The same volume of PDB was similarly treated as a negative control. The flasks were incubated at 25 °C under shaking at 100 rpm for 7 days. Each treatment was done in triplicate and the experiment was repeated twice. From each treatment, an aliquot of mycelia was picked to assess the presence of morphological/structural changes due to the presence of the B. subtilis V26 supernatant on the media by using a light microscope (Olympus Optical Co. LTD) operating with Olympus DP70 camera (magnification 40 × ).

Potato tubers were immersed for 5 min in a 0.5% sodium hypochlorite, and then rinsed several times with sterile distilled water, air-dried at room temperature and finally cut into 2-cm slices. Following a 72-h culture harvesting of B. subtilis V26, the spores and culture supernatant were separated by centrifugation at 8000 rpm for 20 min. Next, the pellet (spores) was washed twice with distilled water and bioassayed by dipping the potato slices into different cell suspensions, namely: 10⁶ spores/mL washed cell suspension (SPO1), 10⁹ spores/mL washed cell suspension (SPO2), 10⁹ spores/mL unwashed cell culture mixture (SPO2-CM), commercial biofungicides Prevam (0.3%, v/v) (ORO Agri, International Ltd. USA), biofertilizer Nitrophoska (NPK: 2 g/L) (COMPO^®) and sterile distilled water as a negative control (dipped for 30 min), prior and 24 h after the inoculation with a 6-mm agar disk of R. solani. The potato slices were sealed in polyethylene-lined plastic boxes to maintain high humidity and incubated at 25 °C, seven days in the dark. The incidence of decay was assessed by measuring the lesion area on each potato slice as compared to the total potato slice surface area using a visual scale from 0 to 4, where 0 = absence of lesion area, 1 = 1–25% potato slice surface with lesion area, 2 = 26–50% potato slice surface with lesion area, 3 = 51–75% potato slice surface with lesion area, 4 ≥ 75% potato slice surface with lesion area. Each treatment was replicated five times and the antagonism assays were repeated twice. The reduction of decay incidence was calculated according to the following formula:

The ability of B. subtilis V26 to protect potato against R. solani infection was assessed on tubers of potato cv. Spunta (highly susceptible to black scurf) [9,13]. Initially, tubers were immersed in 0.5% sodium hypochlorite for 5 min, rinsed in running tap water and air-dried at room temperature. Then, the tubers were subjected to the following treatments during 30 min: 10⁶ spores/mL (SPO1), 10⁹ spores/mL (SPO2), 10⁹ spores/mL culture mixture (SPO2-CM), commercial biofungicides Prevam (0.3% v/v) (ORO Agri, International Ltd. USA), biofertilizer Nitrophoska (NPK: 2 g/L) (COMPO^®) and sterile distilled water as a negative control. Each treatment was replicated five times and the assay was repeated twice. Treated tubers were planted in plastic pots containing sterilized peat (Potgrond H), mixed or not with R. solani inoculum. The pathogen inoculum was obtained from 7-day-old cultures grown on PDA Petri dishes kept at 25 °C, and then it was scraped and blended with sterile distilled water at the rate of five plates per kilogram of peat. After planting, pots were placed in greenhouse for 60 days and irrigated daily to maintain high peat moisture.

Disease incidence and plant growth parameters were determined 60 days after planting. Plants were harvested, washed, and rated for root canker as described by Brewer and Larkin [27] as follows: 0 = no disease symptoms; 1 = brown discoloration of roots; 2 = cankers covering < 25% of the root circumference; 3 = 25–75% roots covered by cankers; 4 = 75% coverage by roots cankers; and 5 = root completely nipped off or death of the plant. The black scurf was assessed on a scale of 0–5, as described by Brewer and Larkin [27]: 0 = no visible sclerotia; 1 = sclerotia covering 1% of the tuber area; 2 = 2–5% of the tuber area affected; 3 = 5–10% of the tuber area affected; 4 = 10–15% of the tuber area affected and 5 or more then 15% of the tuber area affected. The reduction of disease incidence was calculated according to the following formula:

The number, the height and the fresh weight of stems, and the fresh weight of roots were also recorded for each plant at harvesting time.

Data were subjected to analysis of variance using SPSS software. Mean values were compared using Duncan's multiple range test at the 5% (P < 0.05) level of significance.

In dual culture assays of strain V26 against R. solani, a significant inhibition zone of the mycelia growth was shown, suggesting the presence of antifungal metabolites secreted by this strain. The B. subtilis V26 antagonistic effect was evaluated by serial dilutions and its culture supernatant was able to inhibit the growth of R. solani even after dilution to 1:32 (Fig. 1a). The strain V26 was also able to produce chitosanase and proteases (Fig. 1b and c), which may play a role in dissolving and penetrating the cell walls of R. solani, as reported by McQuilken and Gemmell [28].

The antifungal activity of the strain V26 against R. solani was quantified by the incorporation of its culture supernatant in PDA plates. As shown in Fig. 2A, the antagonist V26 exhibited a significant inhibition of the pathogen growth compared to the untreated control. Mycelial growth reduction reached 80% at low percentages of supernatant (3% v/v) in comparison to the untreated control. An increase in the percentage of the V26 supernatant induced a significant increase in fungal growth inhibition. This reduction of mycelial growth caused by the V26 supernatant was also accompanied by morphological alterations of R. solani hyphae (Fig. 2B). Compared to R. solani treated by PDB, the supernatant culture of V26 (1% v/v) caused uncommon hyphae deformation and developed swelling at hyphal tips (Fig. 2B). Moreover, when R. solani hyphae were treated with increased supernatant percentage (9% v/v), hyphal deformation and enlargement of cytoplasmic vacuoles were increased, since its cell wall disintegrated, protoplasm leaked out, and the mycelia cracked (Fig. 2B).

The efficacy of the B. subtilis strain V26 to inhibit the growth of R. solani was estimated on potato slices 24 h before and after being inoculated with R. solani (Fig. 3). The observations from treated potato tuber slices and untreated ones showed differences (P < 0.001) in decay incidence. A slight decrease in the incidence of the disease was shown on the potato slices when the application of the antagonist V26 (SPO1 and SPO2-CM) and of the biofertilizer nitrophoska was made 24 h after inoculating with the fungus (Fig. 3a). Like commercial biofungicide Prevam, V26 (SPO2) treatment significantly decreased the decay incidence in potato slices by only 30% in comparison to the inoculated but untreated control. However, the decay incidence observed in potato slices treated with B. subtilis V26 (SPO1, SPO2 and SPO2-CM), nitrophoska and Prevam 24 h prior to inoculation with the pathogen was significantly different from that developed in the control set treated with R. solani alone. The disease incidence was nearly zero for all V26 treatments compared to untreated potato tuber slices (Fig. 3b). Prevam treatment at (0.3% v/v) gave also a complete control of R. solani infection. However, the Nitrophoska treatment at 2 g/L reduced decay incidence at only 25%, in comparison to the control (Fig. 3b).

In pot experiments, data were collected 60 days after inoculation. The results of V26 biocontrol assays are presented in Figs. 4 and 5A. Disease incidence on roots of all treated potato plants was significantly lower than that of the untreated ones that were used as controls (P < 0.001). V26 (SPO2-CM) treatment resulted in low disease incidence on roots, with a reduction of 63% compared to the untreated one (Fig. 4). Moreover, the disease incidence on potato tuber caused by R. solani was also significantly (P < 0.01) reduced by the different treatments applied when compared to the controls. The application of V26 significantly reduced black scurf in potato tuber compared to the control (Fig. 5Ba), with a decrease in the incidence rate to 81% when using V26 (SPO2) treatment (Fig. 5Bb). No statistical differences were observed between (SPO2) treatment and the application of the commercial biofungicide, Prevam. No significant protection was noted on tubers treated by the biofertiliser “Nitrophoska”, which was shown to be less effective in reducing the incidence of the disease, in comparison to the pathogen control. Compared to the untreated potato plants, the treated ones recorded a significant increase in the growth parameters (Table 1).

In fact, V26 (SPO2-CM) treatment significantly enhanced plant height, with an increase of 37.89%, comparatively to the control. In addition, the number of stems significantly increased more than 70% when potato tubers were treated by V26 (SPO2-CM), in comparison to the untreated control. Shoot fresh weights were significantly increased for all treatments, compared to the untreated control. The improvement in shoot fresh weight of plant growing in presence of V26 (SPO2-CM) is more than 80%, in comparison to the untreated control. V26 treatments also promoted root growth, with a maximal increase of 37.34% reached by SPO2-CM treatment (Table 1). It seems also that the use of the whole culture was the best treatment to promote potato plant growth, followed by spore suspension (Fig. 6).