Chitinolytic Bacillus subtilis Ege-B-1.19 as a biocontrol agent against mycotoxigenic and phytopathogenic fungi Mikotoksijenik fitopatojenik küflere karşı kitinolitik Bacillus subtilis

Background: Biological control of pathogenic fungi is a possible alternate to the chemical control, which is harmful to humans and environment. Soil-borne Bacillus strains can be potential biocontrol agents and a source of lytic enzymes. Aim: This study aimed to examine biocontrol potential and lytic enzyme activities of a soil isolate Bacillus subtilis Ege-B-1.19. Materials and methods: Strain was identified by biochem-ical and 16S rRNA gene analysis and its biocontrol activity was investigated against Aspergillus niger EGE-K-213, Aspergillus foetidus EGE-K-211, Aspergillus ochraceus EGE-K-217, Fusarium solani KCTC6328, Rhizoctonia solani and Colletotrichum gloeosporioides KACC40689. Chitinase, chitosanase, N-acetyl- β -hexosaminidase and protease activities of B. subtilis Ege-B-1.19 were also determined. Chitosanase was purified using Sephadex G-150 column and its molecular weight was determined by SDS-PAGE. Chitooligosaccharides production using chitosanase was carried out and analysed by TLC and HPLC. Results: Results depicted that B. subtilis Ege-B-1.19 has shown inhibitory effects against all the test fungi. Chitinase, chitosanase, N-acetyl- β -hexosaminidase and protease activities were determined as 2.7 U mL −1 , 7.2 U mL −1 , 6.2 U mL −1 and 12.2 U mL −1 , respectively. Molecular weight of purified chitosanase was 44 kDa. Chitosanase hydrolysed chitosan to glucosamine (GlcN), dimers (GlcN) 2 and trimers (GlcN) 3 . Conclusion: Bacillus subtilis Ege-B-1.19 can be anticipated as useful biocontrol agent and its chitosanase can be utilized for enzymatic synthesis of chitooligosaccharides.


Introduction
In the absence of effective chemical control methods and to reduce environmental pollution, the biological control of plant pathogens is currently considered a key practice in sustainable agriculture. Bacillus, Paenibacillus, Pseudomonas and Trichoderma species all express good antagonistic activities under controlled laboratory or greenhouse conditions. Thus, antagonistic microorganisms play a very important role in biological control [1]. In the past 30 years, there have been extensive research activities to explore and develop strategies based on microbial antagonists to biologically control postharvest pathogens [2]. The bacterial biocontrol mechanisms for plant protection against fungal diseases include systemic resistance, parasitism and competition for nutrients. Bacteria present in soil are also known to produce a number of enzymes and antimicrobial compounds [3].
Bacillus species are the most studied antagonist among the soil born bacteria [4]. They have been employed in biocontrol [5,6] and bioremediation [7]. Bacillus species were found to inhibit a number of fungi under in vitro conditions [8,9]. Bacillus strains can secrete various secondary metabolites, which inhibit the pathogenic fungi. It is also known that Bacillus strains are able to produce various hydrolytic enzymes, including chitosanases, chitinases, N-acetyl-β-hexoaminidase, proteases and laminarinases in order to inhibit the growth of pathogenic fungi by degrading their cell wall [8,10,11]. Among these hydrolytic enzymes chitosanase (EC 3.2.1.132) are important enzymes not only for the biocontrol of pathogenic fungi, but also with reference to their ability to produce chitooligosaccharides (COS): a degradative product of chitosan. Chitosan and its derivatives have shown various antibacterial, antifungal, antitumor and antioxidant activities [12] which made them important to be used in several industries including agriculture, food, cosmetics, biocontrol, and wastewater treatment. However, the difficult dissolution of chitosan limits its use in several industries including food and biomedical applications [13]. Unlike chitosan, chitosan oligosaccharides and short chain D-glucosamine units are readily soluble in water. Chemical and enzymatic ways are generally used to synthesize chitosan oligosaccharide, however, chemical methods have some disadvantages such as difficult control of chemical reactions, formation of various secondary compounds and use of harmful chemicals [12,13]. Enzymatic methods including microbial chitosanases showed excellent performances in COS production [14][15][16].
With the aim to discover potential biocontrol agents, this study was designed to isolate chitinolytic Bacillus strains from vineyard soil. Bacillus subtilis Ege-B-1.19 having the highest chitinolytic activity was identified and examined for its in vitro antagonistic activity against phytopathogenic and mycotoxigenic fungi. Furthermore, partial purification of chitosanase from B. subtilis Ege-B-1.19 and enzymatic production and quantitative analysis of COS were also the objectives of this study.

Soil sampling and analysis
Soil samples were obtained as composites from five different sampling points of a vineyard in Manisa province, Turkey (38° 40′N and 27° 03′E and 39° 08′N and 27° 37′E). At each sampling point, firstly, surface material was dug to a depth of 2-3 cm. Soil samples were taken using sterile equipments at a depth of 25-30 cm for chemical analysis and isolation of potential biocontrol agents. The sample soils were analyzed in TARIS R&D Center, Soil Laboratory, Bornova, Izmir for water content, pH, salt (%), carbonate (%), texture, clay (%), sand (%), nitrate-N (%), total phosphorus and potassium.

Isolation of endospore forming bacteria and screening of chitosanolytic activity
Soil samples were suspended in sterile distilled water, shaken vigorously and incubated on a water bath (100 rpm, 80°C) for 30 min to kill all vegetative microbial cells. After incubation, the soil samples were serially diluted (10fold), plated on NA medium and incubated at 30°C for 2 days and spore forming bacteria were isolated. Colonies arising from the isolates survived after heat shock were transferred to a maintenance medium. The isolates were stored in glycerol at −80°C [17,18]. Screening of chitosanase production was carried out using chitosanase detection agar (CDA) containing 1% soluble chitosan, 0.13% sodium phosphate, 0.3% potassium phosphate, 0.05% sodium chloride, 0.1% ammonium chloride, 0.024% magnesium sulfate, 0.001% calcium chloride and 2% agar (pH 6.5). Isolates were inoculated on the plates, incubated at 30°C for 5 days and colonies surrounded by clear zones were isolated [8]. The isolates selected through the screening procedure were then cultured in LB-broth containing 0.2% swollen chitin and 0.2% colloidal chitosan and incubated at 30°C and 200 rpm for 2 days for the measurement of lytic enzyme activities.

Identification of the bacterial isolate
Isolate Ege-B-1. 19, showing highest chitosanolytic activity was selected and performed Phenotypic identification test by using BBL Chrystal Gram Positive ID System Test Kit in accordance with the manufacturer's instructions. Results were interpreted according to BBL™ Crystal™ Identification System. For molecular identification, DNA isolation were performed using the UNIQ-10 Spin Column Genomic DNA Minipreps Kit (SK201&202) by the manufacturer's instructions. Bacterial 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the universal eubacteria primers 8F, 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R, 5′-GGTTACCTTGTTACGACT-3′. Partial 16S rDNA raw sequence data were imported into the BioEdit Sequence Alignment Editor and a contiguous consensus sequence was generated. Using blast against GenBank database (EU099356), the nearest relatives of these consensus sequence were identified. Data obtained after sequencing were submitted in the NCBI GenBank database to attain accession numbers.

Measurement of lytic enzyme activities
Chitinase activity was detected by measuring the amount of reducing sugar released from swollen chitin by the Schale's method using a standard curve for N-acetylglucosamine (GlcNAc) [19]. The assay was performed by mixing 0.9 mL of 1% swollen chitin (pH 6.0) and 0.1 mL of fermentation broth. After incubation for 2 h, at 37°C, chitin hydrolysis reaction was terminated by adding 0.2 mL NaOH (1 N). One millilitre Schale's reagent was then added and the reaction mixture was kept in boiling water for 15 min followed by chilling on ice. The amount of reducing sugars released in the supernatant was measured spectrophotometrically at 420 nm. One unit of chitinase activity was defined as the amount of enzyme required to produce 1 μmol GlcNAc per hour [19].
Chitosanase activity was measured by DNS method using a standard curve of D-glucosamine (GlcN) [20]. The assay was performed by mixing 0.9 mL of 1% soluble chitosan (pH 6.0) and 0.1 mL of fermentation broth. After incubation for 30 min, at 37°C, hydrolysis reaction was terminated by adding 0.2 mL NaOH (1 N) and 1.5 mL DNS reagent. The reaction mixture was kept in boiling water for 5 min followed by chilling on ice. The amount of reducing sugars released in the supernatant was measured spectrophotometrically at 540 nm. One unit of the enzyme activity was defined as the amount of enzyme required to release 1 μmol of reducing sugars per min at 37°C [20].
N-acetyl-β-hexosaminidase activity was assayed by using p-nitrophenyl-N-acetyl-b-D-glucosaminide (p-NP-Glc-NAc) as substrates. The reaction mixture containing 50 μL of 5 mM p-nitrophenyl-N-acetylglucosaminide, 50 μL of crude enzyme, and 100 μL of 50 mM McIlvaine buffer (pH 5.0) was incubated at 45°C for 15 min. The reaction was stopped by adding 1 mL of 0.2 M Na 2 CO 3 and the amount of p-nitrophenol released was determined by measuring the absorbance at 405 nm. One unit of β-N-acetylglucosaminidase was defined as the amount of enzyme required to release 1 μmol of p-nitrophenol per min [21].
Protease activity was measured using 450 μL of 1% azocasein in 0.1 M phosphate buffer (pH 7.0) and 50 μL enzyme solution. Reaction mixture was incubated at 50°C for 30 min. Reaction was terminated by adding 250 μL of 25% (w/v) trichloroacetic acid. Mixture was centrifuged at 10,000 rpm and 600 μL of supernatant was mixed with 600 μL of 1 N NaOH. Enzyme activity was measured at 440 nm and 1 U was defined as the amount that increased absorbance to 0.01 at 440 nm per min [22].

In vitro antifungal activity assay
Isolate Ege-B-1.19 was tested for antifungal activity against A. niger EGE-K-213, A. foetidus EGE-K-211, A. ochraceus EGE-K-217, F. solani KCTC 6328, R. solani KACC 40111 and C. gloeosporioides KACC 40689. Plugs of 6 mm in diameter were cut from the outer edges of the colony from the test fungi and placed in the center of PCA plates. Fermentation broth of isolate Ege-B-1.19 was filtered through 0.22 μm pore size membrane filters and used to prepare filter paper discs. Fifty microliter of filtered fermentation broth was dispensed on sterilized filter paper discs (9 mm) and discs were placed on the PCA plates around the fungal plugs. Fifty microliter of steriled water was used as a control. The plates were incubated for 10 days at 28°C and the ability of the isolate Ege-B-1.19 to inhibit test fungi was observed daily.

Hydrolysis of chitosan by chitosanases to produce chitosan oligomers
For the hydrolysis of chitosan to chitosan oligosaccharides, the reaction mixture containing 1 mL of 1% soluble chitosan, 1 mL of 100 mM sodium acetate buffer (pH 5.5), and 20 μL of culture supernatant was incubated at 37°C for 30 min and the reaction was stopped by immersing the tubes in boiling water for few minutes.

Investigation of chitosan oligomers by analytical methods
Chitosan oligosaccharides were analyzed by TLC on Silica gel 60 (Kieselgel 60 F254; Merck). A solvent system of n-propanol:ethylacetate:ammonia solution:water (6:3:3:1) was used and sugar spots on the plates were visualized by spraying 0.2% ninhydrin into 99% ethyl-alcohol. For the quantitative analysis of chitosan oligosaccharides, HPLC analysis was carried out with a Carbohydrate Analysis column (300 × 3.9 mm) using a solvent system of acetonitrile and water (68:32) at a flow rate of 1.0 mL min −1 with a refractive index detector [20].

Purification of chitosanase from the isolate Ege-B-1.19
The isolate Ege-B-1.19 was grown in LB-CC broth at 30°C, 200 rpm for 48 h. The cells were removed by centrifugation at 6000 g for 20 min. The supernatant was fractionated by addition of 85% saturation of (NH 4 ) 2 SO 4 and precipitates were collected by centrifugation. The precipitates were dissolved in appropriate volume (2 mL) of 50 mM sodium acetate buffer at pH 5.5 followed by overnight dialysis against the same buffer. After dialysis, the enzyme was concentrated with polyethylene glycol (PEG) and eluted on Sephadex G-150 gel column (2.6 × 40 cm) previously equilibrated with the same buffer. The fractions were collected and chitosanase enzyme activity and protein amount were measured [23].

SDS-PAGE for the detection of molecular weight and chitosanase activity
Sodium dodecyl sulfate polyacrylamine gel electrophoresis (SDS-PAGE) was performed with 12.5% polyacrylamide gel containing 0.01% soluble chitosan as a substrate for chitosanase activity. After SDS-PAGE, the gel was incubated at 37°C for 2 h with gentle shaking for renaturation in 100 mM sodium acetate buffer (pH 5.0) containing 1% purified Triton X-100 and 1% skim milk. The gel was washed with distilled water and incubated for another 1 h at 37°C in 100 mM sodium acetate buffer (pH 5.0) with 1% purified Triton X-100. After renaturation, the gel was transferred to Tris-HCl buffer (0.5 M, pH 9.0) containing 0.01% (w/v) Calcofluor white M2R stain, and incubated for 7 min followed by several washes. Chitosanase activity on the gel was visualized under UV light [23].

Characterization of the soil sample
The vineyard soil sample was collected from center of Manisa in the summer season (2004)(2005). Table 1 summarizes the properties of soil samples.

Isolation of endospore forming bacteria and screening of chitosanolytic activity
A total of 28 endospore forming isolates were obtained from the vineyard soil samples and 13 isolates have shown chitosanolytic activity on CDA plates. Isolate Ege-B-1.19 represented the best result by showing the largest clear zone, and therefore, it was selected for further studies.

Identification of Ege-B-1.19 isolate
Ege-B-1.19 isolate was a Gram-positive, rod-shaped and endospore forming bacterium. The cells were 0.8-1.0 μm wide and 2.0-3.0 μm long. Oval shaped, 0.7-0.8 μm wide and 1.0-1.5 μm long spores were produced by this isolate as observed by OLYMPUS (CX-31) model digital camera with microscope (10X40). Ege-B-1.19 isolate was identified as B. subtilis by the BBL™ Crystal™ Identification System which was further confirmed by 16S rRNA gene sequence. Sequence was submitted to GenBank and accession number (EU099356) was obtained. Phylogenetic tree was constructed with MEGA 5.0 by using the Neighbor-joining method with 1000 bootstrap replications ( Figure 1).

Chitosan hydrolysis and analysis of chitosan oligomers
Crude chitosanase was used to hydrolyse soluble chitosan for the synthesis of COS. TLC results showed that the chitosanase had released chitosan oligosaccharides from chitosan, the hydrolysate was primarily containing GlcN, small amount of dimers (GlcN)2 and trimers (GlcN)3. The results suggested that chitosanase from B. subtilis Ege-B-1.19 showed exo-and endo-type cleavage. These results were also confirmed by the HPLC analysis results, and quantitative analysis was done using a standard curve ( Figure 3). Quantitative analysis showed that chitosan hydrolysate was containing 50.6 mg of GlcN, 5.2 mg of (GlcN)2 and 8.2 mg of (GlcN)3.

Partial purification of chitosanase from B. subtilis Ege-B-1.19
An extracellular chitosanase was partial purified from the culture supernatant (1500 mL) of B. subtilis Ege-B-1.19 by Sephadex G-150. As shown in Figure 4, two chitosanase peaks of F1 (major fraction) and F2 (minor fraction) were separated. Specific chitosanase activity of F1 fraction was 4.7 U mg −1 and of F2 fraction was 33.7 U mg −1 . The purification steps were summarized in Table 3. There was 0.3 fold increase in specific activity of the F1 fraction of chitosanase as compared to the fermented broth, while purification fold of F2 fractions was 2.2. The protein obtained from F1 and F2 fractions were subjected to SDS-PAGE and chitosanase enzyme activity was detected by Calcofluor white M2R staining. More than 10 bands of different molecular weights were observed in F1 fraction, while no chitosanase enzyme activity was detected. On the other hand, only one sharp band of chitosanase was observed in F2 fraction (44 kDa) and chitosanase activity was also detected ( Figure 5).

Discussion
Vineyard soil sample from Manisa province (38° 30′N 27° 42′E). Manisa province has mainly 20% clay, 20% sand, 20% sandy-clay, 14% loamy-clay, 9% clay type of soil with a very high organic matter content. Soil texture,  mineralogy, nutrient contents, pH, and organic matter is proposed as the major driver affecting the bacterial community composition [24]. Bacillus species are the most studied antagonist among the soil borne bacteria [4].
Since Bacillus species have the ability to produce spores, therefore, it is advantageous to use them for biocontrol as compared to Gram-negative and non-spore forming bacteria. Endospores are particularly suitable to be used as biocontrol agents, because spores can stay alive for a longer period of time in highly variable environmental conditions (nutrient levels, temperature and water activity). Also many Bacillus spp. are known to produce antifungal antibiotics and extracellular hydrolytic enzymes.
COS, being recognized as low molecular weight and water soluble chitosans, have much greater demand in many fields than that of precursor molecule. COS possess a wide range of biological activities and have numerous promising applications in multiple fields such as medicine, cosmetics, food and agriculture [12]. Chitosanase have the ability to split chitosan into oligosaccharides ranging from dimer to octamers. Many chitosanases from Bacillus strains have shown endo-type cleavage [37][38][39], whereas some chitosanases from fungi such as Aspergillus fumigatus KH-94, Aspergillus oryzae IAM2660 and some bacteria such as Nocardia orientalis IFO 12806, Enterobacter sp. strain G-1 have exo-splitting activity that hydrolyse chitosan to glucosamine [37,40]. In the present study, the catalytic pattern of B. subtilis Ege-B-1.19 chitosanase was examined by using soluble chitosan. TLC analysis showed that the chitosanase had released chitosan oligosaccharides, mainly GlcN, GlcN2, and GlcN3 from soluble chitosan ( Figure 3). Although primary products from chitosan by plenty of chitosanases were di-, tri-and tetra-chitooligosaccharides, B. subtilis Ege-B-1.19 chitosanase hydrolysed chitosan to diand tri-chitooligosaccharides along with the production of high amount of glucosamine. These results suggested that the mode of action of chitosanase produced by B. subtilis Ege-B-1.19 is both exo-and endo type.
Bacillus subtilis Ege-B-1.19 could be a potential biocontrol agent biocontrol agent for vineyard uses. More investigations are necessary to increase the effectiveness of the bacterium before applying the bacterium to the vineyards. The chitosanase of B. subtilis Ege-B-1.19 hydrolysed chitosan into GlcN as well as oligomer, therefore, this chitosanase has shown both exo-and endo type hydrolysis and could be a preferable candidate for many biotechnological application.