Antimicrobial Activities and Antioxidant Properties of Bacillus Mojavensis I4 Lipopeptides and Application in the Biocontrol of Potato Dry Rots Caused by Fusarium Solani

Lipopeptides are diverse metabolites produced by various bacterial and fungal genera known for their antimicrobial and surfactant activities with diverse environmental, pharmaceutical application and also agronomic applications as biocontrol agents. In this study, the presence of NRPS genes in Bacillus mojavensis I4 was conrmed by PCR and this bacterial strain could produce diverse lipopeptides which belonged to fengycin, and surfactin families. The antioxidant activity of I4 biosurfactants was determined through four different in vitro assays. Furthermore, antimicrobial activity assays indicated that I4 lipopeptides exhibited marked inhibitory activity against several bacterial and fungal strains. The I4 lipopeptides were effective in the biocontrol of Fusarium solani that causes potato dry rot disease. Preventive treatment decreased the fungi penetration by almost 80 % after 15 days. This nding suggested promising application of I4 lipopeptides in potato tuber storage. of potato dry rot caused by F. Solani. The results of our work demonstrate that B. mojavensis I4 could produce different type of lipopeptides according to the positive detection of ve NRPS genes. I4 lipopeptides were found to exhibit strong antioxidant and antimicrobial activities Moreover, I4 lipopeptides effectively reduced the potato tuber dry rots caused by F. solani using preventive treatment. Considered together, results obtained here suggested the potential application of I4 lipopeptides in potato tubers treatment before their storage.


Introduction
Potato (Solanum tuberosum L.) ranks fourth among the most important food crops in the world after wheat and rice in terms of world food production and as an energy source (Thompson et al. 2009) According to FAO, potatoes production is estimated about 376 million tons in 2016 and classi ed as the fourth crops production after corn (1060 MT), wheat (794 MT) and rice (740MT) worldwide. Potato tubers, the edible part of the plant, are affected by many plant diseases caused by bacteria, fungi, viruses and nematodes (Toth et al. 2011). Fungal pathogens are the principal causes of crop losses before and after harvest (Strange and Scott 2005). Fusarium solani is one of the main soil-contaminating fungi that causes Fusarium wilt, which is a dry rot of tubers. This fungus penetrates in the potato tubers through wounds and causes drying and hardening. They form cavities lined with whitish mycelia. Dry rot of potatoes causes signi cant losses during storage.
Several effective fungicides had been used against pathogens, but as a long-term solution they were not effective owing to the expense, exposure risk, fungicide residue and environmental hazard concerns (Nguyen et al. 2013). Therefore, the use of biocontrol based on environment-friendly microorganisms represents an attractive alternative for plant disease management. Many research reports are available regarding, the use of Bacillus species as biocontrol agents (Mnif et al. 2015) and their derived biomolecules including lipopeptides to inhibit fungal spore germination (Mnif et al. 2015). Recently, lipopeptides were employed as suitable alternatives to synthetic medicines with lower toxicity, biodegradability and ecological compatibility. Indeed, Antioxidant, antimicrobial as well as antitumoral properties of lipopeptides are equally exploited in cosmetic and pharmaceutical elds (Tabbene et al. 2012). Biosurfactants are composed of 7 or 10 α-amino acids linked to one unique β-amino or β-hydroxy fatty acid with several families such as surfactin, iturin and fengycin (Akpa et al. 2001). Lipopeptides produced by Bacillus genus was synthesized by multi-enzyme templates know as non-ribosomal peptides synthetases (NRPSs). The present study aims to evaluate the antioxidant and antimicrobial activities of the lipopeptides produced by Bacillus mojavensis I4 and prove their potentiality in potato tuber rot biocontrol against Fusarium solani.

Plant material
Potato tubers (Solanum tuberosum L) cv. Spunta produced and stored in our laboratory were selected free of wounds / rots and homogeneous in maturity / size, as much as possible.

PCR detection and cloning of NRPS genes
Five pairs of degenerate primers (Table 1) were used to amplify the genes encoding for surfactin synthetase, fengycin synthetase, bacillomycin synthetase, mycosubtiline synthetase and kurstakine synthetase as previously described (Abderrahmani et al. 2011). The PCR products were excised from Agarose gel and puri ed using the Pure Link Quick gel extraction Kit (Invitrogen) and cloned into the pGEM-T Easy vector (Promega) following the manufacturer instructions. Subsequently, cloned PCR products were sequenced using M13 universal sequencing primers and the obtained sequences was analyzed by BLASTX against the non redundant protein database on NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Tks-R CCACCDKTCAAAKAARKWATC Using IUPAC DNA code: Y = C or T, M = A or C, K = G or T, W = A or T, D = G, A or T, S = G or C, B = G, T or C, R = A or G Antioxidant activity DPPH radical-scavenging capacity The DPPH radical-scavenging capacity of I4 biosurfactants was evaluated as described (Bersuder et al. 1998). Brie y, the aliquots of the different concentrations of the lipopeptides (1 to 10 mg/mL) were mixed with 0.02% DPPH in ethanol. The mixture was shaken vigorously, stood at room temperature for 60 min in the dark and the absorbance was measured at 517 nm. A lower absorbance of the reaction mixture indicated higher DPPH free radical-scavenging activity. BHA was used as positive control and the DPPH radical scavenging capacity was calculated using the following formula: Where A control is the absorbance of the control reaction (containing all reagents except the sample) and A sample is the absorbance of sample reaction (with the DPPH solution). The experiment was carried out in triplicate with similar results and mean values were reported. Ferric reducing antioxidant power (FRAP) assay The ability of I4 lipopeptides to reduce iron was determined as previously described (Yildirim et al. 2001) with slight modi cations. 0.5 mL of I4 lipopeptides at different concentrations (from 1 to 10 mg/mL) were mixed with 1.25 mL of 0.2 M potassium phosphate at pH 6.6 and 1.25 mL of 1% (w/v) potassium ferricyanide solution. The reaction mixtures were incubated for 20 min at 50°C and stopped by the addition of 0.5 mL of 10% trichloroacetic acid. After centrifugation at 2700 g for 10 min, 1.25 mL of the supernatant solution were mixed with 0.25 mL of 0.1% ferric chloride and 1.25 mL of distilled water. After incubation at room temperature for 10 min, the absorbance was measured at 700 nm. The higher reducing power was indicated by the higher absorbance of the reaction mixture. Experiments were done in triplicates.

Iron (Fe 2+ ) chelating activity
For iron chelating activity, 0.1 mL of lipopeptides with a concentration range of 1 to 10 mg/mL was mixed with 0.45 mL of deionized water and 50 µl of 2 mM ferrous chloride. After 5 min of incubation at room temperature, the reactions were initiated by the addition of 200 µl of ferrozine solution (5 mM). The mixture was incubated for 10 min at room temperature and the absorbance measured at 562 nm. Control tube was prepared with the same manner with substituting the lipopeptides by water. EDTA was used as a positive control and the percentage of inhibition of ferrozine-Fe 2+ complex formation was determined as follows: Where A control is the absorbance of the control reaction and A sample is the absorbance of biosurfactants.
Experiments were done in triplicates.
DNA nicking assay DNA nicking assay was evaluated using empty pGEM-T easy plasmid (Promega). A mixture of 10 µL of I4 lipopeptides (1 or 4 mg/mL) and 0.5µg of plasmid DNA were incubated for 10 min at room temperature followed by the addition of 10 µl of Fenton's reagent ( Antimicrobial activity was investigated as previously described (Berghe et al. 1991). A culture suspension (200 µL) of the tested microorganisms (10 8 spores/mL and 10 6 cfu/mL of fungal and bacterial strains, respectively) was spread on potato dextrose agar medium and Luria-Bertani (LB) agar, respectively.
Subsequently, 60µl of lipopeptides solution (20 mg/mL) were added to each well (5 mm diameter), punched in the agar layer, allowed to diffuse and then incubated for 3 hours at 4°C. Cultures were carried for 24 h at 37°C or 72 h at 30°C for bacterial and fungal strains, respectively. The diameters of growth inhibition zones were measured for the evaluation of antimicrobial activity. Moreover, Ampicillin (20 mg/mL) and carbendazim (0.5 mg/mL) were used as positive controls for bacterial and fungal strains, respectively. All tests were done in triplicates and the results were averaged.

Minimum inhibitory concentration (MIC) determination
The minimum inhibitory concentration (MIC) of I4 lipopeptides was determined by liquid growth inhibition assay in a microtiter plate well (Froidevaux et al. 2001). MIC is de ned as the lowest concentration of lipopeptides that completely inhibits the tested strain growth. Indeed, 0.1 mL of cell suspension were distributed in a microtiter plate well and then an equal volume of serial dilution of lipopeptides, made from 20 mg/mL solution, was added. The cells were enumerated on nutrient agar medium before and after incubation at 37°C for 24 h.

Effect of I4 lipopeptides on F. solani mycelial growth and morphology
To evaluate the effect of I4 lipopeptides on the growth of F. solani, 10 ml of PDA medium containing I4 lipopeptides at different concentrations (0.5, 3.0 and 5.0 mg/mL) were spread on Petri dishes (diameter 6 cm). After solidi cation, an agar disk (6 mm) containing the mycelium of F. solani was deposited in the center of each Petri dish. After 10 days of incubation at 30°C, the mycelial growth inhibition (MGI) was calculated according to the following formula: To investigate the effect of I4 lipopeptides on the mycelium morphology, a microscopic observation of mycelium near the zone of inhibition was carried (× 40 magni cations). Non treated mycelium was used as control.

Application of I4 lipopeptides in potato tuber rots biocontrol
To evaluate the capacity of I4 lipopeptides in the biocontrol of F. solani dry rot, potato tubers were surface sterilized in sodium hypochlorite (0.5%) for 10 min, washed using sterile distilled water several times and then dried at room temperature. A volume of 0.1 mL of spore suspension containing 10 7 spores/mL of F. solani was poured into a well perforated in the tubers with a sterile cork borer. The treatment of potato tubers with different concentrations of I4 lipopeptides was evaluated by two different methods. The preventive method which consisted on the addition of 0.1 mL I4 lipopeptides (3 and 5 mg/mL) in the wells 24 h before the infection by fungi while in the curative method the lipopeptides solutions were added at 24 h post infection. Sterile distilled water and the commercial fungicide carbendazim were used instead of I4 lipopeptides as negative and positive control, respectively. Tubers were incubated in sterile plastic bags at 30°C for 15 days at high relative humidity. Meanwhile, the rot development was evaluated by measuring the diameter of F. solani spreading lesions. After treatment, tubers were longitudinally cut and the parameters of dry rot induced (maximal width (w) and depth (d)) were recorded. The penetration of the pathogen into tubers was measured as follow (Lapwood et al. 1984). Where: w width of dry rot (mm) d depth of the dry rot (mm) p depth of the inoculation well (mm)

Statistical analysis
The data reported here were mean values with standard deviation. Mean values of three biological replicates obtained from each experiment were compared using the Tukey's HSD tests. Moreover, Pearson's correlation coe cient was employed to calculate the correlations between data. All analyses were carried using JMP software version 10 (SAS INSTITUTE).

Results And Discussion
Detection of NRPS genes in B. mojavensis I4 PCR was used to detect the presence of NRPS genes in the I4 strain. Positive ampli cation at expected size was obtained for the tested primers suggesting the presence of ve NRPS genes (Fig. 1). BLASTX analysis of each ampli ed NRPS gene sequences ( Table 2) showed that the fragment ampli ed by As1-F/ Ts2-R primers pair showed 91% identity with surfactin synthase gene from Bacillus subtilis, which is characterized as surfactin-producing strains (Duitman et al. 1999). Similarly, ampli ed fengycin DNA fragment (primers Af2-F/ Tf1-R) shared high similarity (95%) with fengycin synthetase from B. subtilis (Table 2). Moreover, PCR fragments corresponding to Bacillomycins, Mycosubtilin and Kurstakin showed signi cant homology with different peptide synthetases with a homology higher than 88% (Table 2). These founding suggested that I4 strain could produce surfactin and fengycin. Indeed, previous HPLC analysis revealed that surfactin is the major compound in the lipopeptides mixture produced by I4 strain

Ferric reducing antioxidant power (FRAP) assay
The reducing powers of I4 lipopeptides was determined and compared to BHA at different oncentrations were showed in Fig. 2b. The ability of I4 lipopeptides to reduce Fe 3+ to Fe 2+ was concentration dependent and the reducing power of I4 lipopeptides increased signi cantly until a concentration of 6 mg/mL beyond which reducing power seemed to be constant. However, the I4 lipopeptides showed lower reducing power activities compared to BHA at the same concentrations. The reductive capacity of I4 lipopeptides reached 2.0 (A700 nm) at a concentration of 8 mg/mL which was is slightly higher than those of Bacillus mojavensis A21 lipopeptides which showed the same reducing activity at a in their structure which act as electron donors such as hydroxyl groups that could react with free radicals to convert them into more stable products (Yalcin and Cavusoglu 2010).

Iron (Fe 2+ ) chelating activity
Iron chelating activity determination showed a dose dependent chelating effect of I4 lipopeptides (Fig. 2c). Indeed, at a concentration of 10 mg/mL, the ferrous-chelating effect of I4 lipopeptides was 76.21% against 98.25% obtained with EDTA (positive control). The IC 50 values for crude biosurfactant and EDTA were estimated at 0.4 ± 0.03 and 0.29 ± 0.01 mg/mL, respectively. This activity was lower than those reported by Zouari et al. (2016b) which found that the chelating effect of biosurfactant of B. subtilis SPB1 and EDTA were 80.32% and 98.89%, respectively, at a concentration of 1 mg/mL and those from Bacillus methylotrophicus DCS1 lipopeptides which exhibited a ferrous-chelating activity of 79.8% at 4 mg/mL (Jemil et al. 2017). This difference in Iron chelating activity observed among different bacillus species could be explained by the structural differences of lipopeptides produced by each species. Indeed, the differences in the sequence of amino acids and carbon atoms in the fatty acids provide biosurfactants with diverse chemical structures and physiochemical properties (Liu et al. 2015).

DNA nicking assay
The DNA nicking assay was used to investigate the scavenging ability of I4 lipopeptides against hydroxyl radical generated by the Fenton reaction. Results (Fig. 2d) showed that the plasmid DNA was completely degraded in the presence of hydroxyl radicals generated by the Fenton reaction (Lane 3) compared to the native plasmid (Lane 4). The migration pro les of the plasmid pre-incubated with the I4 lipopeptides at 1 mg/mL (Lane 1) and 4 mg/mL (Lane 2) is similar to the control plasmid (Lane 4   The effect of I4 biosurfactants on mycelial growth of F. solani Fusarium dry rot is a postharvest disease which caused severe potato tubers losses could be caused by several Fusarium spp among them F. solani remains the most prevalent (Daami-Remadi et al. 2006). In this work we are considering the use of crude I4 lipopeptides as cost effective biocontrol agent of this disease instead of chemical control. The growth inhibition assay showed that the I4 lipopeptides possessed an important antifungal activity against F. solani with an inhibition zone of 24 mm (Table 3). Further, the growth of F. solani were evaluated during 10 days in the presence of different lipopeptides concentrations (0.5, 3 and 5 mg/mL) in comparison to the mock control without biosurfactants. Results showed that the I4 lipopeptides effectively inhibited the growth of F. solani compared to the control (Fig. 3a). To a rm the antifungal potency of I4 lipopeptides, mycelium of F. solani was microscopically observed near the zone of inhibition (Fig. 3b). In fact, the non-treated control showed articulate hyphae with normal mycelium and smooth surfaces (Fig. 3b) while the treated mycelium showed irregular shape with excessive lyses and destructed spores. Moreover, no fungal growth was observed with 5 mg/mL of lipopeptides which suggested total destruction of the spores ensuring high e cacy of antagonistic activity. This observation suggested that I4 biosurfactants acted on the cell surface by the potent permeabilizing activity leading to cellular death. Similarly the cell lysis of the pathogenic fungi F. solani when treated by SPB1 biosurfactants was earlier reported (Mnif et al. 2015).

Application of I4 biosurfactants in potato tuber rots biocontrol
The e cacy of I4 biosurfactants in the treatment of potato tuber rot caused by F. solani was investigated in vivo (Fig. 4). For this purpose, both curative and protective treatments were carried out. Commercial antifungal agent carbendazim was used as positive control (Fig. 4a). Results showed that I4 lipopeptides were effective in the biocontrol of the disease at all tested concentrations in both curative and protective treatments ( Fig. 4a and 4b). Penetration values for controls and the treated tubers revealed that the lipopeptides reduced the penetration of the pathogen up to 80% at 5 mg/mL in preventive treatment while in curative one the reduction was almost 67% (Fig. 4c). Moreover, signi cant differences were observed between the tested concentrations in both the treatments. Indeed, the reduction of the fungus penetration was signi cantly more effective with 5 mg/mL than 3 mg/mL. Besides, at the same concentration (5 mg/mL), the preventive treatment was more e cient than the curative one (P ≤ 0.05). However, lipopeptides from B. mojavensis I4 was less effective compared to the commercial antifungal agent as it reduced 90% of the fungus penetration using the preventive treatment. This result is in accordance with the previous report that preventive treatment was more effective than preventive treatment in the biocontrol of F. solani in potato tuber using biosurfactants from B. subtilis SPB1 (Mnif et al. 2015

Conclusion
In the present study we explored the in vitro antioxidant and antimicrobial activities of lipopeptides from B. mojavensis I4 and its e ciency in the biocontrol of potato dry rot caused by F. Solani. The results of our work demonstrate that B. mojavensis I4 could produce different type of lipopeptides according to the positive detection of ve NRPS genes. I4 lipopeptides were found to exhibit strong antioxidant and antimicrobial activities Moreover, I4 lipopeptides effectively reduced the potato tuber dry rots caused by F. solani using preventive treatment. Considered together, results obtained here suggested the potential application of I4 lipopeptides in potato tubers treatment before their storage.