Rayda Siala, Laboratoire de Génie Enzymatique et de Microbiologie, Université de sfax Ecole Nationale d'Ingénieurs de Sfax, B. P. “1173” 3038 Sfax, Tunisia. E-mail: firstname.lastname@example.org
To investigate the distribution of chitinase IO8 in Bacillus cereus strains, the enhancing effects of the chitinase-producing B. cereus strains on biocontrol potential by dual culture assay and in vivo assay against Botrytis cinerea and also the enhancing effects of the chiIO8 on disinfectant properties against seed-borne diseases. Moreover, the application of chiIO8 treatment was also observed to improve the germinative energy.
Methods and Results
The purification steps included ammonium sulfate precipitation, with columns of DEAE-Sepharose anion-exchange chromatography and Sephacryl S-400 high-resolution gel chromatography. The method gave a 5·8-fold increase in the specific activity and had a yield of 17%. The molecular weight of the partially purified chitinase chiIO8 was found to be around 30 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and optimal temperature of the partially purified chitinase were pH 6·5 and 65°C, respectively. The thermostable chitinase still retained the activity after incubation for 100 min at 65°C, and it was increased about 1·25 times than that of the control (before heating) when the enzyme solution heated at 65°C for 60 min. The partially purified chitinase chiIO8 displays a wide inhibitory spectrum towards all phytopathogenic fungi tested. chiIO8 also exhibited effective disinfectant properties against seed-borne diseases.
The present investigation emphasizes the potential of chitinase-producing micro-organism as promising biocontrol agents of fungal plant pathogens with chitinous cell wall. The novel chitinase chiIO8 proved an efficient, environmentally safe and user-friendly solution.
Significance and Impact of the Study
This is the first investigation devoted exclusively to analyse the distribution of chitinase in B. cereus. It infers that the chitinase produced by B. cereus might play a role in the activity of the biopesticide.
Chitin, the β-1,4-linked polymer of N-acetyl-d-glucosamine, is one of the most abundant polysaccharides in nature, next to cellulose. It is a major cell wall constituent of higher fungi belonging to chytridiomycetes, ascomycetes, basidiomycetes and deuteromycetes, insect exoskeletons and crustacean shells (David 2004). Interestingly, the presence of chitin in the cyst wall of the human pathogen, Entamoeba histolytica, has also been demonstrated (Das et al. 2006). Therefore, chitinases, the hydrolytic enzymes that specifically degrade chitin, are gaining much attention worldwide (Makino et al. 2006; Wang et al. 2006). Chitinases are produced by several bacteria (Ajit et al. 2006; Natarajan and Ramachandra 2010; Ghorbel-Bellaaj et al. 2012), actinomycetes (El-Tarabily et al. 2000), fungi (Viterbo et al. 2001; Nawani et al. 2002) and also by higher plants (Matsushima et al. 2006). These chitinases are used in various applications such as biological control of fungal pathogens (Someya et al. 2001; De la Vega et al. 2006; Chang et al. 2007), preparation of oligosaccharides and N-acetyl d-glucosamine (Makino et al. 2006; Wang et al. 2006) and protoplasts from filamentous fungi (Balasubramanian et al. 2003; Prabavathy et al. 2006; Yano et al. 2006). Micro-organisms produce the chitinase primarily for the assimilation of chitin as a carbon and (or) nitrogen source (Flach et al. 1992; Kupiec and Chet 1998). Chitinases have been isolated from a variety of bacteria including Bacillus spp., and some of them are reported to produce multiple forms of chitinases with different molecular masses (Ajit et al. 2006), although chitinase production was reported in different species of Bacillus such as B. cereus (Ghorbel-Bellaaj et al. 2012), B. licheniformis (Waldeck et al. 2006), B. subtilis (Wang et al. 2006) and B. thuringiensis sub sp. Kurstaki (Driss et al. 2005).
In this study, a chitinolytic bacterium B. cereus IO8 isolated from Tunisian soil was investigated for its potential biocontrol activity against pathogenic fungi. The specific objectives of the study were to (i) use a dual culture plate assay to test the antifungal activity of the isolate against a range of fungal phytopathogens, (ii) test the ability of the isolate to control grey mould caused by Botrytis cinerea on tomato plants, (iii) extract, purify and characterize the chitinase(s) of the B. cereus isolate, (iv) assess the antifungal activity of the purified chitinase against a range of fungal phytopathogens and (v) test the effect of the purified chitinase on germination of tomato seeds.
Materials and methods
Screening of chitin-degrading bacteria
Bacillus cereus IO8 isolated from Tunisian soil showed high chitinase activity on the 0·5% colloidal chitin agar (CCA) medium, obtained using Hsu and Lockwood (1975) method. Primary screening was performed by spot inoculating bacteria on CCA using toothpick heads of 2 mm diameter and incubated at room temperature. The zone of clearance due to chitin hydrolysis was recorded up to 5 days. The bacterial isolate was subjected to secondary screening. It was performed with the culture filtrate of the selected bacteria using well diffusion method. Bacillus cereus IO8 were grown in nutrient broth containing 1% colloidal chitin. One millilitre of bacterial inoculum with 0·5 OD was inoculated to 100 ml of medium and incubated at 100 rpm in a rotary shaker at room temperature. After 2 days of incubation, the culture was harvested and centrifuged at 9630 g for 15 min at 4°C, and the supernatant was collected. Colloidal chitin (0·5%) agar plates were prepared, and wells were made using 9-mm sterile cork borer. Culture filtrate of the isolate was placed at 100 μl in each well and incubated at 37°C. After 12 h, the development of clear zone around the well was observed.
Antifungal activity assay
A dual culture assay was performed to assess the potential biocontrol activity of B. cereus IO8. Antifungal activity was determined against Alternaria solani, Fusarium solani (Olive Tree Institute of Sfax), Fusarium sambucinum, Alternaria citri, Penicillium occitanis, Aspergillus nidulans, Verticillium dahliae and Botrytis cinerea (kindly provided from the culture collection of The Centre of Biotechnology of Sfax). Fungal strains were maintained on potato dextrose agar (PDA) slants, grown at 28°C for 72 h and stored at 4°C. In the dual culture assay, spore solution of B. cinerea (3 × 106 spores ml−1) and bacterial suspension of B. cereus IO8 were streaked on the surface of Luria–Bertani agar at a distance of 4 cm. The growth of both was examined daily for the formation of inhibition zone.
In vivo disease control
Tomato plants of the cultivar Riogrande were transplanted into pots of 12 cm in diameter (one plant/pot) and filled with nonsterilized soil with the following characteristics: 60 g kg−1 clay, 30, 910 g kg−1 and, pH 8·2, electrical conductivity of 1·1 mS cm−l and organic matter content of 1·7 g kg−1, 0·176 g kg−1 total N, 0·042 g kg−1 P and 0·13 g kg−1. After 10 days, fourth groups of 30 plants each were prepared: the first group was kept without treatment and served as control, and the other three groups plant were inoculated with 5 h-pregerminated conidia of B. cinerea (3 × 106 spores ml−1) (1·5 ml plant−1). The pregermination solution contained 6·84 g l−1 sucrose, 1·74 g l−1 K2HPO4 and 10 mg l−1 vitamin B5 at pH 6·3. Three days later, the second group was treated with B. cereus suspension (106 CFU ml−1), the third group was treated with benomyl (6 mg l−1), and the fourth group was treated with distilled water. A 1·5 ml of each solution was applied to each tomato plant using a hand sprayer. The experiment was conducted in three replicates and repeated twice in early spring 2010 and 2011.
The disease severity index of grey mould on the tomato plants was defined as the percentage of leaf area with symptoms, where 0 = no symptoms, 1 = 0·1–5·0%, 2 = 5·1–20·0%, 3 = 20·1–40·0% and 4 = 40·1–100·0% leaf area symptomatic. Disease severity (%) was calculated using the following formula: (R (number of diseased leaves × disease severity index)/(4 × number of leaves rated)) × 100. The disease control value was calculated using the following formula: disease control (%) = [(A–B)/A] × 100, where A is the disease severity on plants inoculated with the pathogen alone, and B is the disease severity on plants inoculated with the pathogen and treated with the bacterial suspension of B. cereus or the fungicide benomyl. After 2 months, the disease severity was scored.
Enzyme production and purification
For the production of chitinase, strain B. cereus IO8 was grown in 100 ml of fresh medium (3% w/v chitin, 0·1% KH2PO4, 0·05% MgSO4·7H2O, 50 mmol l−1 sodium phosphate buffer, pH 7·0) in a 250-ml Erlenmeyer flask at 30°C for 3 days. The culture broth was centrifuged for 20 min at 12 000 g at 4°C. The supernatant was used for enzyme purification. The supernatant was brought to 80% precipitation with ammonium sulfate and left standing overnight at 4°C. The precipitate was collected by centrifugation at 12 000 g (4°C) for 20 min and redissolved in 50 mmol l−1, pH 4·2, sodium acetate buffer. The solution was dialysed for the removal of ammonium sulfate in the same buffer for 12 h at 4°C. The dialysed sample was passed through a DEAE-Sepharose fast flow column (1·6 × 45 cm, Amersham Biosciences, Freiburg, Germany) pre-equilibrated with 50 mmol l−1, pH 4·3, sodium acetate buffer. First, the column was washed with five column volumes of the same eluting buffer, and then, the enzymes were fractionated with a linear gradient of 0–1 mol l−1 NaCl in 50 mmol l−1, pH 4·3, sodium acetate buffer at 0·5 ml min−1 for 400 min. The fractions with chitinase activity were pooled and precipitated again with 80% saturation of ammonium sulfate solution. The mixture was left overnight at 4°C and then centrifuged at the same conditions as stated earlier. The collected precipitates were dissolved in 50 mmol l−1, pH 7·3, Tris-HCl buffer. The obtained solution was applied on a Sephacryl S-400 high-resolution column (1·6 × 60 cm, Amersham Biosciences) pre-equilibrated with 50 mmol l−1, pH 7·2, Tris-HCl. Then, the column was washed with the same buffer at the flow rate of 0·5 ml min−1. The fractions with chitinase activity were collected and dialysed against 50 mmol l−1, pH 7·2, Tris-HCl buffer for the determinations of purity and characterization.
Chitinase activity measurement
A fluorometric assay was used to determine chitinase activity using 4-methylumbelliferyl- N,N′,N′′-chitotriose (Sigma, St. Louis, MO, USA) as a substrate. The amount of 4-methylumbelliferone (4-MU) released was measured spectrofluorometrically using a fluorescence spectrophotometer (excitation 390 nm and emission 450 nm). One unit (U) of chitinase activity was defined as the amount of enzyme required to release 1 μmol of 4-MU per min at 37°C (Haggag and Abdallh 2012). Protein concentration was determined by the Bradford method (Bradford 1976) using bovine serum albumin as standard.
Determination of the chitinase molecular weight
The molecular weight of the chitinase was determined by SDS-PAGE. It was carried out with 12% (w/v) isolation gel and 5% (w/v) concentration gel according to the method of Laemmli (1970). The gel was stained with Coomassie brilliant blue R-250 in methanol–acetic acid–water (5 : 1 : 5, v/v) and decolorized in 7% acetic acid. The following proteins were used as standards: phosphorylase b (97·4 kDa), bovine serum albumin (66·2 kDa), ovalbumin (43 KDa), carbonic anhydrase (31 kDa), trypsin inhibitor (20·1 kDa) and α-lactalbumin (14·4 kDa).
Effects of temperature and pH on chitinase activity
To determine the optimum temperature, chitinase activity was examined at various temperatures (35–80°C) in 50 mmol l−1, pH 6·5, Tris-HCl buffer (10 U ml−1). Thermal stability of the chitinase was measured in terms of residual activity after incubation of the partially purified chitinase at various temperatures ranged from 50 to 70°C for 30–240 min and from 80 to 90°C for 10–60 min. The activity of the partially purified chitinase was also measured at different pH values. The pH was adjusted using the following buffers: 50 mmol l−1 sodium acetate buffer (pH 4·0–5·0), 50 mmol l−1 sodium phosphate buffer (pH 5·5–7·0) and 50 mmol l−1 Tris-HCl buffer (pH 7·5–9·0). The reaction mixtures were incubated at 65°C for 45 min, and the activity of the enzyme was assayed to measure the optimum pH.
Effect of chitinase on the growth of phytopathogenic fungi
Sclerotia or fungal mycelium (2·5 mg) was transferred to 250-ml Erlenmeyer flasks containing 50 ml of 1% potato dextrose infusion and partially purified chitinase at different concentration (0·1–0·8 U). Cultures were incubated at 30°C, 250 rpm for 72 h in triplicate. Fungal growth was determined by dry weight. Cultures without chitinase were used as a control.
Effect of chitinase on seed germination
To investigate the effect of chitinase on the germination of tomato seeds infested with each fungus, plastic trays, measuring 52 × 25 × 4 cm, with 200 wells each, were filled with a sterile potting mix (Gramoflor GmbH & Co.KG, Vechta, Germany) previously autoclaved twice at 121°C for 20 min and mixed with the dried mycelium of each fungi (5–6 mg kg−1). One seed was planted in each well. One millilitre of partially purified chitinase at concentrations of 0·2, 0·4, 0·6 and 0·8 U mg−1 protein was applied to each well. The number of germinated seeds was recorded.
The results were analysed by one-way analysis of variance (anova) and Tukey's test (P < 0·05) using MINITAB version 10.51 (Minitab Inc., State College, PA, USA) for Macintosh. All tests were carried out in triplicate.
Biocontrol potential of B. cereus IO8
The antagonistic activity of B. cereus IO8 against fungi was demonstrated by a dual culture assay and an in vivo assay against B. cinerea. Dual culture of B. cereus and B. cinerea showed that B. cereus IO8 inhibited the growth of B. cinerea in vitro by secreting antifungal compound(s) out of the bacterial cells, as indicated by the formation of inhibition zone. In vivo tests, treatments with B. cereus and benomyl showed disease control values of 96 and 72%, respectively (Fig. 1). The B. cinerea-infected control plants showed 7% disease control over the duration of the experiment, indicating severe grey mould infection on the tomato plants (Fig. 1).
Purification of the chitinase
There were five protein peaks and one chitinase activity peak eluted when the dialysed chitinase was applied on a DEAE-Sepharose fast flow column. The chitinase activity peak was eluted at 0·8 mol l−1 NaCl. The fractions of the chitinase peak on the DEAE-Sepharose fast flow column were collected, precipitated with 80% saturation of (NH4)2SO4, dissolved and dialysed in 50 mmol l−1, pH 7·3, Tris-HCl buffer for 24 h at 4°C and then applied to a Sephacryl S-400 high-resolution column. Only one strong chitinase activity peak was detected. The chitinase activity peak fraction was collected and run in a SDS-PAGE gel. A single band was obtained with a molecular mass of approximately 30 kDa (Fig. 2). The purification procedures of the chitinase secreted by the B. cereus are summarized in Table 1.
Table 1. Purification of chitinase from Bacillus cereus IO8
75·4 ± 1
967 ± 1·9
12·82 ± 0·65
100 ± 0·0
1 ± 0·2
30·6 ± 1·2
631 ± 1·87
20·62 ± 0·78
65 ± 0·15
1·6 ± 0·5
8·9 ± 0·6
487 ± 1
54·71 ± 0·54
50 ± 1·82
4·26 ± 0·4
6·55 ± 0·43
393 ± 1·87
60 ± 0·82
46·8 ± 1·8
4·68 ± 0·7
2·34 ± 0·14
174 ± 1
74·35 ± 0·69
17 ± 1
5·8 ± 0·32
Effect of pH and temperature on chitinase activity
To determine the optimum temperature of the partially purified chitinase, enzyme reactions were performed at various temperatures from 35 to 85°C in 50 mmol l−1 Tris-HCl, pH 7·3, for 45 min, using colloidal chitin as substrate. The chitinase was highly active at 55–70°C but low active in the ranges of 35–55°C and 75–85°C, respectively. The optimum temperature of chitinase was 65°C. When the reaction temperature reached higher than 75°C, there was a sharp decrease, and only 42% of the maximum activity at 85°C was observed (Fig. 3a). For the determination of the thermostability of the purified chitinase, enzyme solution was incubated for 20–120 min at 55–75° C or 10–50 min at 85–95°C. The pH was kept at 7·2. The residual chitinase activity was more than 85% when incubated at 50–70°C for 100 min, whereas complete inactivation was observed when the enzyme was incubated at 75°C for 120 min, 85°C for 50 min and 95°C for 20 min. So, the chitinase can be classified as thermophilic chitinase. It was interesting to know that the thermostable enzyme from B. cereus IO8 was increased about 1·25 times than that of the control (before heating) when the enzyme solution was heated at 65°C for 60 min (Fig. 3b). Studies of the influence of pH on the enzyme activity were also carried out. The residual relative activity of more than 80% was found at pH range from 5·5–8·0. However, the relative activity was observed to be <50% at pH 4·0. The optimum pH for the chitinase activity was found to be 6·5, which suggested that the chitinase was a nearly neutral enzyme (Fig. 4).
Effect of chitinase on phytopathogenic fungi
The partially purified chitinase exhibited a pronounced antifungal activity against the phytopathogenic fungus F. solani. This fungus was inhibited completely (100%) on PDA plates containing chitinase at a concentration of 0·3 U mg−1 chitinase. Growth patterns at lower concentrations showed that the response of the fungus to the enzyme to be concentration-dependent (Fig. 5).
Addition of 0·6 and 0·8 U chitinase produced a significant (66%) increase in the germination rate (P <0·05) (Fig. 6). The antifungal activity of chitinase was also investigated using eight different strains of phytopathogenic fungi. The purified chitinase showed a broad spectrum of antifungal activity that was effective against all fungi tested (Fig. 7). The effect of chitinase on the germination of tomato seeds infested with those fungi was followed up. All fungi tested reduced the percentage of germination of infested seeds to <35%, with P. occitanis and A. nidulans being the most aggressive, exerting a total inhibition of germination (Table 2). Seeds protected with chitinase showed a very good response for germination. This ranged from 45% for seeds infested with Alternaria citri to 85% for seeds infested with F. solani.
Table 2. Germination of tomato seeds infested with different phytopathogenic fungi in the absence and presence of Bacillus cereus chitinase. Enzyme concentration was 0·8 U mg−1 chitinase
Germination of tomato seeds (%)
8 ± 0·4
80 ± 1·2
35 ± 1·3
45 ± 1
33 ± 1
69 ± 0·8
5 ± 0·2
85 ± 1
13 ± 0·7
68 ± 1·4
0 ± 0·0
75 ± 1
0 ± 0·1
61 ± 0·9
Plant disease caused by phytopathogenic fungi is a problem of increasing concern to farmers and growers in the Tunisian Valley. Therefore, it becomes very timely to launch intensive efforts in searching for promising biological control agents against plant diseases. The present investigation evaluated the potential of B. cereus IO8 isolated from Tunisian soil to control plant pathogenic fungi. This study dealt with chitinase-producing Bacillus as a promising mechanism that could be utilized as biological control agents, because chitin is a major constituent of the fungi. Therefore, production of the chitinase was used as the main criterion for the selection of potential biocontrol agents against phytopathogenic fungi. The significance of chitinase-producing indigenous bacteria in the biological control process was demonstrated through several procedures to ensure their potential to inhibit mycelium growth of phytopathogenic fungi by dual culture assay as well as to reduce the incidence and severity of the disease in vivo assay. In this study, the B. cinerea inoculation experiments showed that compared to the conventional pesticide benomyl, B. cereus IO8 was highly effective in the control of grey mould on tomato plants. This finding confirms earlier findings by El-Tarabily et al. (2000) who reported chitinase-producing bacterial activity against phytopathogenic fungi in vitro and in vivo. This proves importance of the chitinase-producing bacteria as biocontrol agents.
The development of a four-step purification procedure allowed to the partial purification of the novel chitinase, termed chiIO8. The reliability of each purification step was demonstrated by a significant increase in the specific activity of the chitinase, as previously reported with other chitinase (Dahiya et al. 2005). The partially purified chiIO8 was characterized and evaluated for its activity and stability under different conditions as well as for its effectiveness in terms of antifungal action. ChiIO8 was found to be with a molecular mass of 30 kDa. A considerable variation in the molecular weight of chitinase had been earlier reported: 55 kDa for Sanguibacter antarcticus KOPRI 21702T (Park et al. 2009), 47 kDa for Penicillium sp. LYG 0704 (Lee et al. 2009), 72 kDa for B. licheniformis SK-1(Kudan and Pichyangkura 2009), 70 KDa for Streptomyces RC1071 (Gomes et al. 2001), 36 kDa for Aeromonas sp. DYU-Too7 (Lien et al. 2007), 53 KDa for ASCHI53 and 61 KDa for ASCHI61 form Aeromonas schubertii (Liu et al. 2009). Generally, the molecular weight of chitinase was ranged from 20 to 80 kDa. Sometimes, several chitinases were secreted from one strain, whereas there are visible variation in their molecular weight and enzyme activity (Sakai et al. 1998).
The optimum temperature of chitinase ChiIO8 is 60°C. Similar studies were described for other thermophilic chitinase. For example, the optimum temperature of chitinase from Microbispora sp. V2 was 60°C (Nawani et al. 2002) and from Aeromonas sp. DYU-too7 (Lien et al. 2007), Pseudomonas aeruginosa K-187 (Wang and Chang 1997), Cellulomonas flavigena NTOU 1 (Chen et al. 1997) and Bacillus sp.BG-11 was 50°C (Bhushan and Hoondal 1998). ChiIO8 was also observed to be stable up to 60°C for 60 min, whereas other heat-stable chitinase activities such as a novel thermostable chitinase from Thermomyces lanuginosus SY2 (Guo et al. 2008) and a thermostable chitinase from Planococcus rifitoensis strain M2-26 (Essghaier et al. 2009) were found to be decreased at high temperature for a long time. The only research (Purwani et al. 2004) in the currently available reports also found nearly the same results: a relative activity of chitinase enzyme produced by Bacillus sp.13.26 was reported to be 300% after incubation of the enzyme at 65°C for 3 h.
The optimum pH for the chitinase activity was found to be 6·5, which suggested that the chitinase was a nearly neutral enzyme. Similar optimum pH was reported for other chitinase, such as Enterobacter SP. G-1 (Park et al. 1997) and Ps. aeruginosa K-187 (Wang and Chang 1997), whereas the chitinase from T. lanuginosus SY2 (Guo et al. 2008) had the highest activity at pH 4·5, Aeromonas sp. DYU-too7 (Lien et al. 2007) and Bacterium Ralstonia sp. A-471 (Mitsuhiro et al. 2005) at pH 5·0, Microbispora sp. V2 (Nawani et al. 2002) at pH 3·0, B. cereus at pH 5·8 (Wang et al. 2001), B. circulans No.4·1 at pH 8·0 and Beauveria bassiana at pH 9·2 (Suresh and Chandrasekaran 1999).
Interestingly, when compared to their control counterparts, the tomato seedlings treated with chiIO8 (0·8 U mg−1) showed higher and average germination rates. The application of a chiIO8 (0·8 U mg−1) treatment was also observed to improve the germinative energy. In fact, germinative energy can play an important role in the achievement of quick and uniform seedling emergence and the reduction in damping-off incidences, thus improving the yield. Gupta et al. (1999) reported that the use of fungicides is effective in enhancing germination, emergence and growth as well as in reducing damping-off. In addition, accelerated germination is reported to help improve stress resistance and enhance overall plant growth and productivity (Pattan et al. 2001). This could account for the usefulness of the partially purified chitinase for seed disinfection.
Considering the promising properties of the novel chitinase chiIO8, field trials are now in progress to further evaluate the biocontrol potential. The determination of the NH2-terminal amino acid sequence and the study of the chitinase structural genes as well as its regulatory elements are currently underway in our laboratory. This would require a further investigation into the structure–function relationship using molecular study and 3-D structure determination.
The present investigation emphasizes the potential of chitinase-producing micro-organisms as promising biocontrol agents of fungal plant pathogens with chitinous cell wall. The novel chitinase chiIO8 proved an efficient, environmentally safe and user-friendly solution that (i) provides protection against pathogens that attack germinating seeds and emerging seedlings; (ii) aids in the production of healthy vigorous crops and higher yields; and (iii) offers a cheap, safe and easily employed agent that cause no harm to the seed, the plant, the environment and the human being.
This work was supported by the Ministry of Agriculture and Water Resources and the Ministry of Higher Education and Scientific Research, Tunisia.