Microbiol. Res. (2001) 155,309-314 http://www.urbanfischer.de/journals/microbiolres
Antifungal activity of chitinases produced by some fluorescent pseudomonads against Colletotrichum falcatum Went causing red rot disease in sugarcane R. Viswanathan I, R. Samiyappan 2 I
Plant Pathology Section, Sugarcane Breeding Institute, Indian Council of Agricultural Research, Coimbatore 641007, India Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore 641003, India
Accepted: May 5, 2000
Abstract Chitinase production and growth of certain fluorescent pseudomonads isolated from sugarcane rhizosphere on different subtrates were studied. When chitin was substituted for glycerol in King's B medium, 3 of the 4 strains showed enhanced bacterial multiplication. Bacterial cells grown on chitin-containing medium showed enhanced antifungal activity against Colletotrichum falcatum Went causing red rot disease in sugarcane. Chitinase production was significantly higher when chitin was amended to King's B medium. Higher chitinase production was also recorded when fluorescent pseudomonad strains were grown in the medium containing crab-shell chitin. Cell-free bacterial culture filtrate from chitin-containing medium significantly inhibited mycelial growth of the pathogen. These cell-free conditioned media contained 3 to 7 polypeptides. Western blot analysis revealed five isoforms of chitinase with molecular masses of 47,36,32, 20 and 18.5 kDa. A possible role of chitinases in red rot disease management is discussed. Key words: sugarcane - Colletotrichum fa/catum - fluorescent pseudomonads - chitinase
Introduction Fluorescent pseudomonads belonging to plant growthpromoting rhizobacteria (PGPR) are known to enhance plant growth and reduce severity of many fungal disCorresponding author: R. Samiyappan e-mail:
[email protected] 0944·5013/01/155/04-309
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eases (Hoffland et al. 1996; Wei et al. 1996). These PGPR employ different mechanisms to arrest the fungal growth. Production of lytic enzymes like chitinase and ~-l ,3-glucanase by the PGPR strains is considered a major antagonistic property of the strains. These lytic enzymes act on chitins and glucans, which are the major constituent of the cell wall of the majority of fungi except the oomycetes group. Hydrolytic action of these enzymes results in degradation of the fungal cell wall. Antifungal effects of chitinase produced by many biocontrol agents like Trichoderma spp. and Pseudomonas spp. on plant pathogens have been reported (Haran et al. 1996; Krishnamurthy 1996; Krishnamurthy et al. 1999). Red rot disease caused by the fungal pathogen Colletotrichum falcatum Went (Perfect state: Glomerella tucumanensis (Speg.) Arx & Muller) is a threatening disease of sugarcane, causing severe yield loss in most of the sugarcane-growing states in India (Alexander and Viswanathan 1996). Management strategies to reduce the severity of the disease under field conditions have not yielded satisfactory results (Viswanathan et al. 1997). We have isolated certain native strains of fluorescent pseudomonads from the sugarcane rhizosphere; and they were assessed for their effect on red rot disease development in sugarcane. Some of the isolated bacterial strains were found effective in inducing systemic resistance against red rot disease of sugarcane (Viswanathan and Samiyappan 1997, 1999). Further studies were carried out to assess the antifungal properties of chitinases produced by the sugarcane native rhizosphere bacteria. The results of these studies are reported in this paper. Microbiol. Res. 155 (2001) 4
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Material and methods Isolation of bacterial cultures. Native sugarcane rhizosphere strains were isolated from different parts of Tamil Nadu State. Rhizosphere soils adhering to the roots were collected and put into 250 ml conical flasks containing sterile water and kept in a shaker for 15 min. For the isolation of endophytes, internal stalk tissues were cut into small pieces and a suspension was prepared as for rhizosphere soil. A loopful of the suspension was later streaked onto King's B medium (King et al. 1954). The plates were incubated for about 48 h at 27°C. The fluorescent colonies were viewed under UV light at 366 nm. Bacterial growth on chitin-amended media. Selected strains of PGPR were grown on medium containing combinations of chitin (1 %), peptone (2%) and glycerol (1 %) with continuous shaking for 48 h at room temperature (28 ± 2°C). Colloidal chitin was prepared from crab shell chitin (Sigma) as per the method of Berger and Reynolds (1958) and was used throughout the study. Cell wall materials from the fungal pathogen C. falcatum were purified (Chet and Huttermann 1980) and used in certain studies. Later, one ml of medium was taken and the cells were collected by centrifugation at 6000 rpm for 5 min and were resuspended in one m1 of 0.1 M phosphate buffer. For the preparation of colloidal chitin the crab shells were powdered and digested with concentrated HCl (overnight, 4°C). The digested chitin was washed repeatedly with distilled water to adjust the pH to 7.0. The chitin suspension in water was centrifuged, the pellet collected and dried. The fungal cell wall preparation was obtained from the fungal mycelial mats grown on oats broth for 8 days at room temperature. After washing with sterile water, the mycelium was homogenized in chloroform and methanol (1 : 1 ratio). The resulting suspension was filtered and homogenized in acetone. The mycelium was washed with distilled water repeatedly to remove excess acetone and dried at 45°C. The dried chitin and cell wall materials were used at 1% in the medium. The population of the bacteria was assessed and calculated spectrophotometrically at 595 nm (Thompson 1996). Antagonistic activity ofbacterial strains grown on chitin medium against C. falcatum. The fluorescent pseudomonad strains were tested for their antagonistic action against C. falcatum by the dual plate method. The oats agar medium was allowed to settle for one h in sterile Petri dishes. Then an eight-mm mycelial disc (8 days growth) of the pathogen was placed at the centre of the plate and the bacterial strains were streaked on three sides 48 h later. The mycelial growth was recorded at different intervals. At least three replications were maintained for each strain. 310
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Chitinase production on different media. The colorimetric assay of chitinase was carried out as per the procedure developed by Boller and Mauch (1988). The reaction mixture consisted of 10 III of 1 M sodium acetate buffer (pH 4.0), 0.4 ml supernatant from culture filtrate and 0.1 m1 colloidal chitin (10 mg). One h after incubation the resultant chitin oligomers were treated with snail gut enzyme (helicase). Finally, the mixture was incubated with 2 ml of dimethyl amino benzaldehyde (DMAB) for 20 min at 37°C and the absorbance was measured at 585 nm. N-acetylglucosamine (GlcNAc) served as the standard in this assay. The enzyme activity was expressed as nmoles GlcNAc equivalents/min/mi. Antifungal properties of chitinase from fluorescent pseudomonads. The different bacterial strains were grown in medium containing colloidal chitin and C. falcatum cell wall preparation 1% each for 48 h. The culture filtrates were obtained after centrifugation at 6000 rpm for 10 min. The antifungal activity of the potential chitinases in the culture filtrate was assessed as C. falcatum mycelial growth inhibition in Petri plates. About 200 III of culture filtrate (passed through bacteria-proof filters) was placed in an 8-mm well in the oats agar medium, which had been seeded with C. falcatum conidia 48 h before. The treated plates were incubated at 28 ± 2°C. Sterile water was used in place of culture filtrates in the control treatments. The inhibition zone formation was assessed at different intervals up to 96 h. Protein separation by SDS PAGE and Western blotting. The supernatant from the chitin medium was used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970). The protein content of the sample was determined by the Bradford method (Bradford 1976). Hundred Ilg of protein for each sample was loaded into the wells of polyacrylamide gels. SDS-PAGE was carried out in slab gels containing 4% stacking gel and 12% separating gel using the Sigma-Aldrich Techware system (Sigma, USA) with molecular weight markers (Genei, India). The gels were stained with 0.2 % Coomassie brilliant blue (R 250). After SDS-PAGE the proteins were electroblotted onto 0.45-llm nitrocellulose membranes (Sigma, USA) as described by Gallagher et al. (1995). The electrophoretic transfer of proteins was carried out from gel to membrane overnight at 14 V (constant voltage) in a cold room. The membrane was blocked for 1.5 h at room temperature (28 ± 2°C) in Tris-buffered saline (TBS) containing 0.05% Tween and 2.5% w/v gelatin. The membranes were then incubated for 3 h in the diluted primary antibody (barley chitinase antiserum, a generous gift from Dr. S. Muthukrishnan, Professor of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA) in TBST (1 : 3000 dilution). The
membrane was then incubated in affinity-purified goat anti-rabbit immunoglobulin (lgG) alkaline phosphatase conjugate (Sigma, USA) for 3 h. After each incubation, the membranes were washed with TBST thrice for 10 to 15 min each time to remove the unbound antibody. Immunological reaction was visualized by soaking the membranes in alkaline phosphatase colour development reagent.
Results Bacterial growth on different media showed higher growth for all strains in peptone + chitin or peptone + glycerol + chitin. However, for the medium with chitin
and salts a significantly lower population was recorded, indicating the necessity of other sources for bacterial growth (Table I). Bacterial strains grown on different media showed enhanced fungal mycelial growth inhibition in the presence of chitin in the medium for the strains ARR 1 and KKM I but not for the strain VPT4 (Table 2). Chitinase activity in the culture medium was higher when the bacterial strains were grown in the medium containing chitin with salts or chitin with peptone. Among the strains, KKM I was found to produce the highest amount of chitinase in the medium. The normal medium containing glycerol, which is used for mass multiplication of fluorescent pseudomonads, showed significantly lesser chitinase activity (Table 3). Mycelial growth was inhibited significantly in the treated plates
Table 1. Growth of fluorescent pseudomonad strains on different media. Nutrient Chitin alone Peptone alone Peptone + chitin Peptone + glycerol Peptone + chitin + glycerol Mean
Bacterial growth cfu/ml (x 108) ARRI
KKMI
VPT4
VPT9
0.987 1.530 8.383 4.807 8.287 4.799
1.533 6.793 6.793 5.400 5.190 7.657
0.8367 3.5300 2.5330 5.7100 5.6867 3.6570
0.420 2.261 2.465 2.571 2.585 2.060
Media contained MgS04 and K2 HP0 4 (1.4 gil). Values are the mean of three replications.
Table 2. Effect of chitin in the growth medium on the inhibition of C. falcarum mycelial growth by fluorescent pseudomonad strains. Nutrient
Peptone alone Peptone + glycerol Peptone + chitin Peptone + chitin + glycerol Chitin alone Untreated control Mean
Bacterial strains ARRI
KKMI
VPT4
21.00 34.67 12.67 31.67 36.00 89.33 37.56
15.67 9.67 17.33 11.33 14.00 87.33 25.89
65.33 21.00 68.33 70.67 70.00 90.00 64.22
For details see Table I.
Table 3. Chitinase activity of the bacterial strains grown in chitin-containing medium. Nutrient
Peptone alone Peptone + chitin Peptone + glycerol Peptone + chitin + glycerol Chitin alone Mean
Bacterial strains ARRI
VPT9
VPT4
KKMI
8.37 8.47 7.67 7.30 9.47 8.26
6.90 7.37 7.37 7.93 8.01 7.52
6.93 7.73 6.73 9.57 7.53 7.70
8.13 7.77 7.60 10.03 10.67 8.84
Values are given in nmoles of GIcNAc equivalents/min/m\. For details see Table I. Microbiol. Res. 155 (200 I) 4
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Fig. 3. Detection of bacterial chitinases of strain KKM I grown on chitin + cell wall medium by Western blot. I. KKM I and 2. Marker.
Discussion
Fig. 2. Extracellular protein profile of Pseudomonas strain KKMI grown on different media (12% SDS-PAGE). I. Marker, 2. King's B, 3. chitin + peptone, 4. chitin + cell wall, 5. cell wall and 6. chitin.
after 24-96 h, whereas in the control treatments the well was overgrown after 24 h (Fig. 1). Separation of the extracellular protein of the bacterial strain KKM 1 grown on colloidal chitin and fungal cell wall medium showed at least 7 polypeptides with molecular masses ranging from 14 to 65 kDa (Fig. 2). Western blot analysis of conditioned media using chitinase antiserum detected 47, 36, 32, 20 and 18.5 kDa chitinase (Fig. 3). 312
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Chitinases are widely distributed in nature and play an important role in the hydrolysis of ~-I,4 linkages of the GIcNAc polymer chitin, a structural polysaccharide present in different organisms mainly arthropods and fungi (Cabib 1987). Chitinases are produced by a number of bacteria and fungi living in chitin containing habitats such as soil, sediments and marine environments (Gooday 1990). In plants chitinases are present constitutively and are induced systemically also upon treatment with biotic as well as abiotic inducers. Chitinases along with ~-1 ,3-glucanases impart resistance against a wide array of plant pathogens (Mauch et ai. 1988; Young and Hwang 1994). The present study reveals that fluorescent pseudomonad strains belonging to PGPR are able to utilize chitin for growth. Growth of the bacterial strains was better in chitin-containing medium for three of the four strains (Table 1). This indicates that the bacterial strains were able to degrade the complex chitin polymer for their growth. The bacterial strains produced chitinases for the degradation of chitin. Of the three strains tested for antagonistic activity, ARR 1 and KKM 1 showed higher mycelial growth inhibition. Incidentally, these two strains showed higher chitinase production in liquid cultures. Enhanced chitinase activity was recorded when the bacterial strain was grown in chitin-containing medium (Table 3). The enhanced chitinase activity was responsible for the higher inhibition of C. faicatum
mycelial growth (Table 2). Frandberg and Schnurer (1998) recently reported that chitinolytic bacteria from airtight stored cereal grain had strong inhibitory effects on moulds like Aspergillus spp. and Penicillium spp. They also found that addition of 0.15 % chitin to trypticase-glucose extract medium resulted in higher chitinase activity and enhanced mycelial growth inhibition. Gooday (1990) found that Pseudomonas spp., Bacillus spp. and Streptomyces spp. are chitinolytic. Chitinolytic activity of Bacillus amyloliquefaciens, Streptomyces thermoviolacecus, Serratia marcescens, B. circulans, Streptomyces viridificans, and Flavobacterium sp. were reported (EI-Aassar et at. 1992; Tsujibo et al. 1992; Gupta et at. 1995; Frandberg and Schnurer 1998). Watanabe et at. (1990) reported induction of six chitinases in B. circulans grown in YNB medium containing 0.2% chitin. Similarly, fungal antagonist Trichoderma viride MNT7 produced seven chitinase isoforms in chitin-containing medium (Krishnamurthy, 1996; Krishnamurthy et at. 1999). They also found Rhizoctonia solani mycelial growth inhibition by purified chitinases from T. viride. The present study indicates that extracellular proteins produced by fluorescent pseudomonads in chitin-containing medium inhibited fungal growth significantly (Fig. 1). The secreted chitinase may be responsible for this inhibition of the mycelial growth. Enhanced efficacy in biocontrol agents was reported when the strains were transformed with chitinase genes (Samiyappan et at. 1993, 1994). The chitinase genes from plants and microbes have attracted plant protection scientists either to improve the host plant resistance against fungal pathogens or to improve the efficiency of biocontrol agents. Further studies are required on the purification and characterization of the potential chitinase isoforms for further exploitation of the bacterial chitinases. Such studies would help in exploiting useful genes from the fluorescent pseudomonad bacterial strains for the management of red rot disease in sugarcane.
Acknowledgement The work was supported by Council of Scientific and Industrial Research, New Delhi.
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