ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ, 2014, том 50, № 2, с. 184-188
UDC 576.8
MASS SPECTROMETRY IDENTIFICATION OF ANTIFUNGAL LIPOPEPTIDES FROM Bacillus sp. BCLRB2 AGAINST Rhizoctonia solani AND Sclerotinia sclerotiorum
© 2014 S. Elkahoui*, N. Djebali**, I. Karkouch*, A. Hadj Ibrahim*, L. Kalai*, S. Bachkovel*, O. Tabbene* , F. Limam*
*Laboratory of Bioactive Substances, Centre of Biotechnology of Borj Cedria, Hammam-Lif2050, Tunisia **Laboratory of Molecular Physiology of Plants, Centre of Biotechnology of Borj Cedria, 2050, Tunisia
e-mail: elkahoui@yahoo.fr Received March 14, 2013
This work aims to characterize the bioactive molecules produced by an antagonistic Bacillus sp. strain BCLRB2 isolated from healthy leaves of olive tree against Rhizoctonia solani and Sclerotinia sclerotiorum. The bacterial strain isolated showed a high and persistent antifungal activity against the two pathogens. The free-cell supernatant showed also a high antifungal activity against R. solani and at a lower extent against S. sclerotiorum. The partial purification of the antifungal substances with methanol gradient applied to C18 column binding the Bacillus BCLRB2 culture supernatant showed that the 20% and 60% methanol fractions had a high and specific activity against S. sclerotiorum and R. solani, respectively. The mass spectrometry identification of the compounds in the fraction specifically active against S. sclerotiorum revealed the presence of ba-cillomycin D C16 as a major lipopeptide. The fraction specifically active against R. solani contained bacillo-mycin D C15 and 2 unknown lipopeptides. The 80% methanol fraction had a moderate and a broad spectrum activity against the two pathogens and consisted from two iturin D (C13 and C14) as a major lipopeptides.
DOI: 10.7868/S0555109914020081
Rhizoctonia solani and Sclerotinia sclerotiorum are among the most nonspecific, omnivorous pathogens of several crops worldwide. They are able to infect over 200 plant genera species including several important economic crops such as cereals, cabbage, potato, and legumes [1]. Crop rotation is a necessary practice for minimizing many diseases. However, it is not an effective for the control of Rhizoctonia and Sclerotinia diseases because of their broad spectrum. The use of chemical fungicides to fight against these pathogens did not give enough satisfaction because they produce sclerotia which are highly resistant conservation structures which can survive several years in the soil. In addition, these pathogens can survive on alternate wild plants in absence of the crop [2]. In certain case the use of chemical fungicides was successfully in reducing the infection level in crops [3], but chronic treatment with these chemicals may lead to the emergence of resistant fungal strains. Moreover, resistant cultivars are essentially nonexistent [4]. Djebali and Belhassen [3] found that all cultivated potato varieties in Tunisia are susceptible to R. solani attack.
In our laboratory we are interested in searching biocontrol agents against these fungal pathogens [5— 7] and in finding new natural drugs for the treatment of several diseases [8]. The screening of many bacterial strains isolated from soil, marine biofilms and plants showed that the main active bacteria belong to the genus Bacillus [6, 7, 9, 10]. Antagonistic bacteria of the
Bacillus genus are among the most used biological agents to fight against many plant pathogens [11] including the genera Rhizoctonia [12] and Sclerotinia [13]. The biocontrol potential of Bacillus sp. is based upon their ability to produce a broad array of powerful antibiotic compounds, among which lipopeptides [14] from the fengycin, iturin, and surfactin families can be found [15-17].
The aim of this study was to identify by mass spectrometry the antifungal lipopeptides produced by Bacillus sp. strain BCLRB2 against R. solani and S. sclerotiorum.
MATERIALS AND METHODS
Culture conditions of fungi and bacteria. The fungal isolates of R. solani and S. sclerotiorum were grown on potato dextrose agar (PDA, Pronadisa, Spain) medium at 25 °C in the dark and stored on the same medium at 4°C.
The Bacillus strain BCLRB2 was isolated from healthy leaves of Chemlali olive tree (Olea europea L.) in the olive orchard in the Borj Cedria Technopark (Tunisia) in 2009. The leaf surface was disinfected in 70% ethanol solution for 1 min and then in 3% sodium hypochlorite solution for 5 min. The disinfected leaves were washed several times with sterile distilled water, surface dried, cut in small pieces and putted on Luria-Bertani agar medium (g/L): tryptone — 10, yeast ex-
MASS SPECTROMETRY IDENTIFICATION OF ANTIFUNGAL LIPOPEPTIDES
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tract - 5, NaCl - 10, agar - 20 (pH 7.0 ± 0.2). The agar plates were incubated at 30°C for 24 h. Bacterial colonies showing different morphologies were selected and purified on LB agar plates. Bacterial cultures were kept at —80°C in LB liquid medium supplemented with 25% (v/v) glycerol.
Molecular identification of the bacterium. The 16S
rRNA gene of the bacterium was amplified using primers fD1 (5'-AGAGTTTGATCCTGGCTCAG-3') and rD1 (5' - AAGCTTAAGGAGGTGATCCAGCC- 3') according to Mhamdi et al. [18]. The following cycling conditions were used: initial denaturation at 95°C for 3 min; 35 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 2 min; and final extension at 72°C for 5 min. The amplification product was purified from agarose gels using the Promega (USA) PCR purification kit (Wizard SV gel and PCR clean-up systems). Nearly full length 16S rRNA gene was sequenced by Macrogen, Korea. Sequences were assembled by the CAP program available on the Infobiogen site (http://bio-info.hku.hk/services/menuserv. html/) and checked manually. The FASTA program was used for sequence similarities in DNA databases. Nucleotide sequences were aligned using Clustal W software [19]. The nucleotide sequence data was deposited to the GenBank and an accession number was obtained.
In vitro antibiosis tests. The dual culture test. The in vitro antagonistic assay was performed according to the dual culture method on PDA medium. A fungal agar disk (6 mm) isolated from one day old culture was disposed at the center of Petri dishes and the bacterium strain was deposited at 2 cm distance. The antagonistic activity of the studied bacterium strain was estimated by the inhibition of the fungal growth in comparison to a solely cultivated fungal agar disk. The radius (in cm) of the fungal colony in front of the bacterium culture was measured after 7 days of cultivation at 25°C and 16 h photoperiod. The plates were then kept in the incubator at the same conditions for 30 days to determine the persistence of the activity. This bacterium strain was tested in 3 different plates against each fungal pathogen and the experiment was repeated twice.
The cell-free supernatant activity test. The bacterium culture was centrifuged in Falcon tubes for 10 min at 5000 x g and 4°C. The culture supernatant was recovered, freeze-dried and concentrated to dryness using Christ Alpha 2—4 LSC lyophilizer (Germany). The crude extract was dissolved in distilled water at 50 mg/mL, in which the paper disks were dipped, dried under sterile conditions and put on PDA plates inoculated with the fungal agar disk. The plates were incubated at 25 °C and the diameter of the inhibition zone was evaluated after 7 days. The plates were then kept in the incubator at the same conditions for 30 days to determine the persistence of the activity. This experiment was carried out twice.
Partial purification of bioactive substances. For the
partial purification of the antifungal substances, the lyophilized cell-free supernatant was adsorbed on Sep-Pak cartridges C18 column and was eluted with methanol gradient 0, 20, 60, 80 and 100%. The antagonistic activity of the obtained fractions was tested by the paper disk method as described above.
Mass spectrometry analysis. Mass spectrometric analysis was accomplished on an Applied Biosystems MDS Sciex 3200 triple quadrupole mass spectrometer equipped with an electrospray ionization interface. The fraction was dissolved in 30% acetonitrile added with 0.1% formic acid. The mass spectrometer was turned individually by direct infusion of each fraction at a concentration of 20 ppm with a flow rate of10 ^L min-1 using a Hamilton syringe pump (USA). The mass spectrometer was operated in the positive ionization mode. The apparatus was calibrated with peptides of known molecular mass in the 1-2.5 kDa range (PepMix1, LaserBiolabs, France) and in typical measurements, mass accuracy was ±5 ppm. The data acquisition software used was Analyst 1.5.1.
RESULTS
Identification of the antagonistic bacterium strain.
Classical taxonomic findings showed that bacteria isolated from olive trees belonged to Gram-positive bacilli (data not shown). The genomic bacterial DNA was used as a template to amplify a 638-bp PCR fragment encoding the 16S rRNA. The sequencing of the PCR product and the similarity searches against bacterial databases revealed that the bacterial strain isolated (BankIt1559523 seq1 JX501772) belonged to the genus Bacillus. Indeed, the 16S rRNA sequence was 100% identical to B. amyloliquefaciens (GU181234.1, GU936313.1 and HM030988.1) and 99% identical to B. subtilis (FJ908706.1 and GU586140.1) by BLASTN. Based on the partial 16S rRNA gene sequence, the strain BCLRB2 could not be identified as one of the two closely related Bacillus species.
In vitro antifungal activity of Bacillus sp. The dual culture essay on PDA medium showed that the strain BCLRB2 of Bacillus sp. had a high antagonistic effect against R. solani and S. sclerotiorum mycelia growth (Figure). The inhibition region around the bacterium had persisted over one month of dual culture against R. solani (Figure). In addition, this bacterium strain showed a very high antagonistic activity against 3 Fusarium species (F. graminarium, F. oxysporum and F. oxysp
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