MATERIALS AND METHODS

Fungicidal activity. The in vitro antagonistic assay has been carried out against M. laxa, M.

fructicola, M. fructigena, A. niger and P. expansum following the contact-phase method (El-shafie et al., 2012). Fungitoxicity was represented as the growth inhibition percentage (GIP) according to the formula of Zygadlo et al. (1994) [equation 1]. The obtained results were statistically analysed using statistical Package for the Social Sciences (SPSS).

GIP (%) = 100 X (GC - GT)/GC, (1) ………….[equation 1];

where: GC = average diameter of mycelium growth in PDA (control); GT = average diameter of mycelium growth in treated-PDA.

Biofilm formation assay. The ability of B. mojavensis to produce a biofilm was screened using two different media: i) supplemented Luria-Bertani Casamino acid (S-LBC); ii) Minimal Mineral (MM) in 96-well microplate as explained by Conway et al. (2002). The formation of a biofilm was indicated by measuring the absorbance at λ 540 nm using the Microplate reader instrument (DAS s.r.l., Rome, Italy). This assay was repeated twice with three replicate per each

±SDs. The obtained results were statistically analysed using statistical Package for the Social Sciences (SPSS).

Gas Chromatographic Analysis

Bacterial broth culture. The studied bacterium was grown in liquid Minimal mineral (MM) medium adjusted at 10⁸ UFC/ml for 5 days at 24°C under shaker (180 rpm/min).

Extraction of metabolites. The bacterial broth was centrifuged, filtered, lyophilized, and then stored at –20°C. 300 mg of lyophilized aliquots have been extracted using a cartridge syringe (Strata C18-T). The purified filtrate was dissolved in methanol and injected in GC-MS for identification the principal bioactive compounds.

GC-MS analysis. A qualitative analysis has been carried out by a direct injected into HP6890 plus gas chromatograph equipped with a Phenomenex Zebron ZB-5 MS capillary column (30 m x 0.25 mm ID x 0.25 μm film thickness). The chromatograms obtained from the total ion current were integrated without any correction for co-elutions and the results were expressed as percent

68 of the total area of peaks. All peaks were identified from their mass spectra by comparison with spectra in Wiley 6N and NIST 11 libraries.

RESULTS

Antifungal activity. The obtained results demonstrated in Figure (1) showed high significant antagonistic activity against P. expansum. Whereas, moderate inhibition was observed against M. laxa, M. fructigena and M. fructicola. No antagonistic activity against A. niger.

Biofilm formation. Results revealed that the tested isolate was able to form a biofilm in two different nutrient media SLB and MM (Fig. 2). In addition, the biofilm formation ability was also assessed after 24, 48, and 72 hrs of the incubation at 37°C. In particular, results showed a high significant variation of the biofilm formation between the tested media. The substantial attached growth of the studied bacterium in case of SLB media was significantly higher than MM media where the growth in SLB was ranged between 0.792 to 1.081 nm compared to 0.195 to 0.287 nm in case of MM (Fig. 2).

GC-MS analysis. Using direct injection, 17 compounds, accounting for the 58.95% of the total bacterial extract, were identified. The following five constituents were identified as principals substances produced by B. mojavensis: 2-Furanmethanol (3.53%), 1,3-Dihydroxyacetone dimer (17.69%), Benzene-acetaldehyde (2.57%), 4H-Pyran-4-one, 2,3-dihydro-3,5-di hydroxy-6-methyl (16.29%), and 5-Hydroxy-methylfurfural (19.15%) (Table 1).

DISCUSSION

The antagonistic effect of B. mojavensis may correlated to the production of active substances which might have an indirect antagonism via stimulation the host resistance response (Blacutt et al., 2016).

1,25±0.75 1,1±0.19

0,51±0.34

24 hrs 48 hrs 72 hrs b

bc b

c

a

0 30 60 90

M. laxa M. fcola M. fgna A. niger P. exp

GROWTH İNHİBİTİON %

Figure 2. Antagonistic fungal activity of B.

mojavensis. Bars with different letters indicate means values significantly different at P < 0.05 (Tukey post hoc). Data are mean of 3 replicates ± SDs.

Figure 1. Biofilm formation of B.

mojavensis in S-LBC media. Data are mean of 3 replicates ± SDs.

69 Table 1. The main constituents of B. mojavensisusing GC-MS

In particular, the antimicrobial effect of B. mojavensis could be related to furanmethanol such as furfuryl alcohol, furfural and furoic acid as reported also by Chai et al. (2013). Further earlier investigations reported by Dodd and Stillman (1944) have highlighted the antibacterial effects of some furan derivatives compounds such as furan, 2- furaldehyde, 2-furfuryl alcohol and 2-furoic acid against both gram positive and gram negative organisms and concluded that, nitrofuran compounds have bacteriostatic action against both gram positive and gram negative organisms and less activity against Pseudomonas aeruginosa. On the other hand, some oxygenated derivatives of aldehydes such as trans-2-hexenal and benzeneacetaldehyde have promising antimicrobial effects against several phytopathogens as reported by Yang et al. (2015). Some other lipopeptides from Bacillus such as surfactin, iturin and fengycin, are amphiphilic membrane-active biosurfactants and peptide antibiotics with potent antifungal activities. Results showed also that the biofilm formation can be induced by modifying the nutrient media components especially in the case of rich media S-LBC which has accelerated the bacterial growth rate and enhance the formation of a biofilm.

CONCLUSION

The bioactive metabolites produced by B. mojavensis could have promising applications in organic agricultural and biopesticides industry. Biofilm formation of B. mojavensis gives an advantage by providing protection from antimicrobial agents as well as increase its resistance to antibiotic and in this case the biological activities of the studied bacterium could be more effective in controlling other phytopathogens.

REFERENCES

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Blacutt, A. A., Mitchell, T.R., Bacon, C.W. and Gold, S.E. 2016. Bacillus mojavensis RRC101 Lipopeptides Provoke Physiological and Metabolic Changes During Antagonism Against Fusarium verticillioides, Mol. Plant Microbe Interact. 29, 713–723.

https://doi.org/10.1094/MPMI-05-16-0093-R

Chai, W.M., Xuan Liu, Yong-Hua Hu, Hui-Ling Feng, Yu-Long Jia, Yun-Ji Guo, Han-Tao Zhou, and Qing-Xi Chen, 2013. Antityrosinase and Antimicrobial Activities of Furfuryl Alcohol, Furfural and Furoic Acid, Int. J. Biol. Macromol. 57, 151– 155.

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Peak Name Probability Abundance (%) of total

1 2-Furanmethanol 95 3.53 %

3 1,3-Dihydroxy-acetone dimer 64 17.69 %

5 Benzeneacetaldehyde 94 2.57 %

9 4H-Pyran-4-one, 2,3-dihydro-3,5-di hydroxy-6-methyl-

91 16.29 %

13 5-Hydroxymethylfurfural 94 19.15 %

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71 ORGANİK ELMA YETİŞTİRİCİLİĞİNDE SEYRELTME İÇİN KULLANILAN DOĞAL