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Determination of antifungal activity of Pseudomonas sp. A3 against Fusarium oxysporum by high performance liquid chromatography (HPLC)

P. Velusamy1, H. S. Ko2, K. Y. Kim2

Department of Biotechnology, School of Bioengineering, SRM University, Chennai-603 203, India Division of Applied Bioscience and Biotechnology, Chonnam National University, Gwangju 500-757, Korea

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2

ABSTRACT

It has frequently been reported that chitinolytic soil bacteria, in particular biocontrol strains, can lyse viable fungal hyphae and thereby release potential substrates for bacterial growth. The present work was carried out with an objective to get a better understanding of the relationship between chitinolytic and antifungal properties of bacteria that occur naturally in coastal soils, i.e. without artificial selection. Among the bacterial, strain A3 was identified as Pseudomonas sp. A3 based on morphologic observation and 16S rRNA analysis. Strain A3 exhibited a maximum chitinase production of 1.44 U/ml in CC broth after 3 days of cultivation. Besides having chitinolytic activity, the molecular weight of the crude enzyme was estimated to be 56 kDa by SDS-PAGE and zymogram. In vitro assays revealed that the crude chitinase inhibited activity of Fusarium oxysporum as identified by dual plate assay and microscopic methods. Hydrolysis products of the fungal cell wall by the crude enzymes of Pseudomonas sp. A3 were analyzed by high-pressure liquid chromatography (HPLC) and identified as oligosaccharides, which included monomers (GlcNAc), dimers (GlcNAc)2, and trimers (GlcNAc)3 using chitin oligomer standards. The crude chitinase isolated from strain A3 can be directly applied for suppressing growth of viable fungal hyphae. Keywords: Antifungal activity, Pseudomonas, Fusarium oxysporum, chitinase, fungus, 16S rRNA, Zymogram, HPLC

Agric. Food Anal. Bacteriol. 1: 15-23, 2011

InTRoduCTIon

Biological control of plant pathogens by soil bacteria is a well established phenomenon and chitinase production has been shown to play an important role in the suppression of various diseases (Chernin et al., 1995; Hong and Hwang, 2005; Hoster et al., 2005). Chitin (C8H13O5N)n is an unbranched long

Received: September 3, 2010, Accepted: November 26, 2010. Released Online Advance Publication: March 1, 2011. Correspondence: P. Velusamy, [email protected] Tel: - +91-44-22127331 , Fax: +91-44-22121155

chain polymer of glucose derivatives, composed of ß-1,4 linked units of the amino sugar N-acetyl-Dglucosamine (NAGA), which is speculated to play a vital role in fungal defense against toxic stresses. The interest in chitin degrading enzymes and their application in management of fungal pathogens are significant. Chitinases (EC 3.2.1.14), a group of antifungal proteins, catalyse the hydrolytic cleavage of the ß-1,4-glycoside bond present in the biopolymers of N-acetyl-D-glucosamine, mainly in chitin.

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Antagonistic bacteria are considered as ideal biological control agents that mediate one or several mechanism of disease suppression. Among these, hyper parasitism relies on chitinase for degradation of the cell walls of fungi (Chet et al., 1990). The soil-borne Enterobacter agglomerans IC1270 has a broad spectrum of antifungal activity and secretes a number of chitinolytic enzymes, including two Nacetyl-ß-D-glucosaminidases and chitinase. Its biocontrol activity has been demonstrated with Rhizoctonia solani in cotton using Tn5 mutants deficient in chitinolytic activity (Chernin et al., 1995). Hence, chitinolytic enzyme might be considered to have an important role in biological control of fungal pathogens. It has frequently been reported that chitinase producing microorganisms, in particular biocontrol strains, can lyse viable fungal hyphae, thereby releasing substantial level of oligomers and other substances (Cohen-Kupiec and Chet, 1998; Dahiya et al., 2006; De Boer et al., 2001). In the present work, we report a new strain Pseudomonas sp. A3 possessing strong chitinolytic activity, which exhibited an antagonism toward F. oxysporum. Moreover, the antiungal activity of the crude chitinase from strain A3 was also partially characterized.

Agricultural Culture Collection, Suwon, Korea) grown on potato dextrose agar (PDA) medium containing 0.5% colloidal chitin at 30°C for 7 days.

Bacterial identification

To identify the bacterium, a polymerase chain reaction (PCR) was performed to amplify the 16S rRNA gene from the genomic DNA of strain A3 using universal primers fD1 (5'-GAGTTTGATCCTGGCTCA-3') and rP2 (5'-ACGGCTACCTTGTTACGACTT-3') as described earlier (Weisburg et al., 1991). The PCR product was cloned in a pGEM-T easy vector (Promega, Madison, WI, USA). The nucleotide sequence of the 16S rRNA gene was determined by an ABI Prism 377 DNA sequencer (PE Applied Biosystems, Foster City, CA, U.S.A) and compared with published 16S rRNA sequences using Blast search at Genbank data base of NCBI (Bethesda, MD, USA).

Chitinase assay

For determination of chitinase activities, strain A3 was grown in CC broth at 30°C, and samples were taken at 1, 2, 3, 4, and 5 days. Each sample was centrifuged at 8000 ×g for 5 min and the supernatant was used for enzyme activities. Chitinase activity was determined by incubating 1 ml of culture supernatant with 1 ml of 1% colloidal chitin in a 0.05M phosphate buffer, pH 7.0 at 37°C for 1 h. After centrifugation of reaction mixture, the amount of N-acetyl-d-glucosamine released in the supernatant was determined by the standard method (Lingappa and Lockwood, 1962) using N-acetyl-d-glucosamine (GlcNAc) as a standard. GlcNAc present in 0.5 mL of aliquot of supernatant was determined by adding 0.1 ml of K2B4O7 and then boiled for 3 min in a water bath. The tubes were cooled and 3 ml of p-dimethylaminobenzaldehyde was added. Absorbance was read within 10 min at 585 nm against the blank prepared with distilled water without the enzyme present. One unit of chitinase is defined as the amount of enzyme which releases 1 M N-acetyl-d-glucosamine per hour under the conditions of the study.

MATeRIAlS And MeThodS Screening of bacteria

Five soil samples were obtained from different sites of the coastal soils enriched with crab shells in Buan area, Korea. Soils were serially diluted with sterile water until a dilution of 106 colony forming units (CFU) g-1 of soils, inoculated on colloidal chitin (CC) agar medium containing 0.5% colloidal chitin, 0.2% Na2HPO4, 0.1% KH2PO4, 0.05% NaCl, 0.1% NH4Cl, 0.05% MgSO4 7H2O, 0.05% CaCl2 2H2O, 0.05% yeast extract and 2% agar, and incubated at 30°C for 3 days. Strains exhibiting a clear zone (degradation of chitin) around the colony were picked and further subjected to antifungal activity against F. oxysporum f. sp. cucumerinum (KACC 40032, Korean

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Preparation of crude enzyme

Strain A3 was cultured in a 2000 ml Erlenmeyer flask containing 1000 ml of CC broth at 30°C for 3 days in a shaking incubator (180 rpm). After centrifugation of the broth culture at 8000 ×g for 30 min, ammonium sulfate was added to the supernatant at 50% saturation, and the mixture was left overnight at 4°C. The precipitate was centrifuged at 12 000 ×g for 30 min and the pellet was resuspended in 50 mM TrisHCl buffer [pH 8.0], and dialyzed against the same buffer for overnight. The dialyzate was concentrated by lyophilization, and the concentration of protein was determined using bovine serum albumin (Sigma Chemical Co., St. Louis, MO, USA) as the standard (Bradford, 1976).

ed as control. In order to determine the deformation of hyphae, the experiment was carried out at varying conditions such as pH, temperatures, incubation time, and different buffers.

Hydrolysis of fungal hyphae

To determine the hydrolysis of F. oxysporum by crude chitinase (as described in the section on microscope analysis) after 24 h, the reaction was stopped by addition of 200 l 1 M NaOH. The reaction product was centrifuged at 6000 ×g for 30 min, and the supernatant was passed through a 0.22 m membrane filter (Nalgene, Rochester, NY, U.S.A). The enzyme hydrolysate was analyzed by high-performance liquid chromatography (HPLC). The HPLC was performed with acetonitrile:water (70:30, v/v) as the mobile phase at the flow rate of 1 m/min and detected at 210 nm with NH2P50-4E column (Shodex, Tokyo, Japan) (Kuk et al., 2005). The retention times for the peaks obtained in the crude samples of hydrolytic products were compared with the chitin oligomer standard.

Electrophoresis

The concentrated enzyme sample was subjected to electrophoresis in 12% SDS-PAGE, according to the method described previously (Laemmli, 1970). Subsequently, zymogram was demonstrated by copolymerizing 0.01% of glycol chitin (Sigma) in SDSPAGE for the detection of chitinase activity (Trudel and Asselin, 1989).

ReSulTS And dISCuSSIon Isolation and identification of antagonistic bacterium

In our pilot scale screening, various microbial colonies were able to degrade chitin on CC agar medium. Among these, a bacterial isolate that exhibited the maximum halo zone around the colonies, was designated to be the strain A3. Subsequent antimicrobial activity was examined through dual plate assays using various phytopathogens. Interestingly, strain A3 exhibited a strong antifungal activity against F. oxysporum (Fig. 1). From the morphologic observation, strain A3 was found to be a Gram-negative, rod-shaped and polar-flagella bacterium with permissive temperature ranging between 20 ºC to 37 ºC with an optimum at 30 ºC. The genomic DNA of the strain A3 was amplified with universal primers and 16S rRNA gene sequence was analyzed. Alignment of this sequence (1464 bp) through matching

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Antifungal activity

The crude chitinase was assayed for antifungal activity against F. oxysporum by well diffusion assay on a PDA plate. A fungal plug (6 mm diameter) was removed from the 5 day old culture. The plug was transferred onto the center of the PDA plate, which had been loaded with chitinase in the right well and the left well was loaded with the same volume of buffer. The plate was incubated for 5 days at 30ºC and was monitored for a zone of inhibition around the well. However, the antifungal effect was also observed by a light microscope (Nikon, Tokyo, Japan). Two milliliters of the F. oxysporum suspension with crude chitinase (final concentrations of 2.5 U/ml) in 50 mM of sodium acetate buffer [pH 6.0] was added into a 12 well plates (12 mm, Corning, NY, USA). A mixture of hyphae suspension and buffer was treat-

Agric. Food Anal. Bacteriol. · www.AFABjournal.com · Vol. 1, Issue 1, 2011

Figure 1. Inhibition of the growth of F. oxysporum by Pseudomonas sp. A3 on potato dextrose agar (PDA) medium containing 0.5% of colloidal chitin at 30ºC for 7 days.

strain A3 was identified as a member of Pseudomonas sp, and designated as Pseudomonas sp. A3 (Genbank accession number EU784845). Members of the genus Pseudomonas are ubiquitous in soil microorganisms. They are believed to serve as a promising group of biocontrol agents and have been widely evaluated with the production of chitinases (Ajit et al., 2006; Choi et al., 2006; Fogliano et al., 2002; Folders et al., 2001; Neiendam and Sorensen, 1999).

Determination of chitinolytic activity

Strain A3 was investigated for the production of extracellular chitinase in CC broth by spectrophotometry. At 24 h intervals, aliquots of cell cultures were taken, and the chitinase activity was determined by a standard method. The results from culture filtrate of strain exhibited maximum chitinase activity of 1.44 U/ml after 3 days of cultivation and gradually decreased thereafter (Fig. 3). with reported 16S rRNA gene sequences in the Genbank showed high similarity (99 to 100%) to Pseudomonas sp. The phylogenetic tree determined by the Neighbor-joining method showed that Pseudomonas sp. and P. aeruginosa were most closely related to strain A3 (Fig. 2). On the basis of these results,

SDS-PAGE and zymogram

The molecular weight of crude chitinase was determined by gel electrophoresis using a standard marker (iNtRON Biotech, Inc., Gyeonggi-Do, Korea).

Figure 2. Phylogenetic location of strain A3 based on 16S rRNA sequences by Neighbor-joining method program. Phylogenetic tree based on 16S rRNA sequences displaying the relationship between

strain A3 and that of the other species. Reference species with accession numbers were obtained from Genbank databases. Bar indicates 0.10 nucleotide substitutions per site. Pseudomonas otitidis isolate WL15 (EF687744) Pseudomonas sp. M11 (EU375657) 97 97 98 Pseudomonas aeruginosa strain CMG860 (EU037096) Pseudomonas sp. G3DM-81 (EU037286) Strain A2 99 0.10

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Pseudomonas aeruginosa strain MML2212 (EU344794)

Pseudomonas sp. AHL 2 (AY379974)

Figure 3. Determination of chitinolytic activity by Pseudomonas sp. A3 in CC broth medium at 30ºC for 7 days. Mean values were 3 replicates. Bars represent standard error.

Chitinase Activity (units/ml)

Period of Incubation (days)

Figure 4. SDS-PAGE and zymogram of crude chitinase from culture supernatant of Pseudomonas sp. A3 by ammonium sulfate precipitation.

Lanes 1 and 2 were obtained from 12% SDS-PAGE of Coomassie brilliant blue R-250 where lane 1 is molecular weight (MW) of standard markers in kilodaltons, and lane 2 is crude chitinase sample. Lane 3 indicates zymogram demonstrated by copolymerizing 0.1% of glycol chitin in 12% SDS-PAGE. The chitinase activity was detected by visualization under UV light.

The crude samples of strain A3 revealed several bands on 12% SDS-PAGE and the chitinase activity were identified as 56 kDa by zymogram (Fig. 4). The molecular weights of bacterial chitinases ranged from 20,000 to 120,000, with little consistency. Different molecular masses have been reported for other bacterial chitinases as well (Jung et al., 2002; Wang and Chang, 1997).

MW (kDa)

144 84 63

1

Lane 2

3

Relationship between chitinase production and F. oxysporum suppression by Pseudomonas sp A3

The inhibitory effect on the growth of hyphae of F. oxysporum was investigated under different concentrations of crude chitinase by a well diffusion assay on a PDA plate and a concentration of 50 l (2.5 U/ml) yielded the maximum inhibition against F. oxysporum. However, in the control well, the same volume of sodium acetate buffer did not inhibit the pathogen (data not shown). Microscopic observation revealed a morphology of hyphae that appeared as swelled, fragmented and distorted in the wells treated with the enzyme, whereas the hyphae from the control were normal and intact without any distortion (Fig. 5). The results presented here support antibiosis as the mechanism of antagonism against F. oxysporum by strain A3 mediated through chitinase production. Several studies have demonstrated that

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56 kDa

45

34

26.5

Agric. Food Anal. Bacteriol. · www.AFABjournal.com · Vol. 1, Issue 1, 2011

Figure 5. Morphological study of the hyphae of F. oxysporum in sodium acetate buffer supplemented with crude chitinase at a concentration 2.5 U/ml incubated at 40 °C for 24 h. (A) Control: hyphae of F. oxysporum + buffer, (B) Treatment: hyphae of F. oxysporum + crude chitinase

chitinases of potential biocontrol strains can cause deformation of viable hyphae and result in inhibition of hyphae of the test fungi (Fogliano et al., 2002; Giambattista et al., 2001; Mathivanan et al., 1998). When the crude chitinase was further assayed for antimicrobial activity against various microorganisms. As listed in the Table 1, the percentage of inhibitory effect of crude chitinase against R. solani and D. bryoniae were recorded to be approximately 50% and the remaining microorganisms were not significant.

Table 1. Inhibitory effects of crude chitinase against various microorganisms by in vitro dual plate assays

Name of Organisms

Bacteria Pectobacterium carotovorum subsp. carotovorum KACC 10057 Xanthomonas oryzae pv. oryzae KACC 10378 Fungi Phytophthora capsici KACC 40483

Inhibition Ratio*

ND

-

Analysis of antifungal activity

Fusarium wilt is a widespread plant disease caused by many forms of the soil-inhabiting fungus F. oxysporum. Several attempts have been made to exploit the biological control of F. oxysporum by chitinase producing bacteria (Chung and Kim, 2007; De la Vega et al., 2006; Giambattista et al., 2001). To investigate the antifungal effect of crude chitinase, samples of treated hyphae of F. oxysporum was analyzed by HPLC. As shown in Fig. 6, various products of chitin oligosaccharides such as monomers (GlcNAc), dimers (GlcNAc)2, and trimers (GlcNAc)3 were identified using chitin oligomer standards. Other peaks that were detected may have belonged to chitosan or glucan oligomers. However, there was no detectable number of peaks found in control. Yet, in the present study, chitinolytic bacterium strain A3 may have an important role in the hydrolysis of fungal hyphae and release of substantial

+ ++ ± ++

Didymella bryoniae KACC 40900 Botrytis cinerea KACC 40573 Rhizoctonia solani KACC 40117 Colletotrichum gloeosporioides KACC 40689

-

*Antimicrobial effect of crude enzyme 50 l (2.5 U/ ml) was assayed by well diffusion assay on agar medium. The percentage of inhibition of growth was calculated from the mean values as: % Inhibition = (A-B)/A x 100, where A = microorganism growth in control, and B = microorganism growth in chitinase. The inhibition was reported as (ND) for any undetected inhibition of growth from below 5%, (-) between 5% and 15%, (±) between 15% and 25%, (+) between 25% and 35%, (++) between 35% and 50%. Triplicates were run simultaneously to obtain each value.

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Figure 6. HPLC chromatograms of hydrolysis products from the hyphae of F. oxysporum and crude chitinase mixture after 24 h of incubation at 40 °C. [A] Chitin oligomer standard (GlcNAc)n, [B] Hydrolytic

products of crude sample obtained from hyphae of F. oxysporum + crude chitinase. Peak numbers are referred as 1- monomer (GlcNAc) ; 2- dimer (GlcNAc)2 ; 3- trimer (GlcNAc)3 ; 4- tetramer (GlcNAc)4 ; and 5- pentamer GlcNAc)5.

(1) [A] (2) (3) (4) Standard (GlcNAc)n

(5)

[B]

Hyphae + Crude chitinase

(2) (1) (3)

Retention Time (min.)

Agric. Food Anal. Bacteriol. · www.AFABjournal.com · Vol. 1, Issue 1, 2011 21

levels of chitin oligomers. A previous study reported that chitinase ChiA71 from Bacillus thuringiensis subsp. pakistani completely hydrolyzed colloidal chitin to GlcNAc monomers after incubation for 24 h (Thamthiankul et al., 2001). More recently, Van et al. (2008) suggested that chitinases from Trichoderma aureoviride DY-59 and Rhizopus microsporus VS-9 could release different oligosaccharides after hydrolysis from the hyphae of Fusarium solani.

ConCluSIon

From the results presented in this experiment, a positive correlation can be inferred between the production of chitinase and suppression of the growth of F. oxysporum. However, it is necessary to study the secretion of other lytic enzymes as well, especially cellulase, ß-1,3-glucanase, and laminarinase, as chitinase may combine with other lytic enzymes to exhibit synergism, and result in high levels of antifungal activity. Therefore, we suggest that Pseudomonas sp. A3 may be an optimal candidate for use as a biocontrol agent of Fusarium wilt in tomato, but further studies are needed to evaluate more extensively this possibility.

ACknowledgeMenT

This study was supported by the Korean Research Foundation - second stage of BK21, and National Research Laboratory (NRL) Program from the Ministry of Science and Technology (MOST), Korea.

RefeRenCeS

Ajit, N. S., R. Verma, V. Shanmugam. 2006. Extracellular chitinases of fluorescent pseudomonads antifungal to Fusarium oxysporum f. sp. dianthi causing carnation wilt. Curr. Microbiol. 52:310-316. Bradford, M. M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.

Chernin, L., Z. Ismailov, S. Haran, I. Chet. 1995. Chitinolytic Enterobacter agglomerans antagonistic to fungal plant pathogens. Appl. Environ. Microbiol. 61:1720-1726. Chet, I., A. Ordentlich, R. Shapira, A. Oppenheim. 1990. Mechanism of biocontrol of soil-borne plant pathogens by rhizobacteria. Plant Soil. 129:85-92. Choi, G. J., J. C. Kim, E. J. Park, Y. H. Choi, K. S. Jang, H. K. Lim, K. Y. Cho, S. W. Lee. 2006. Biological control activity of two isolates of Pseudomonas fluorescens against rice sheath blight. Plant Pathol. J. 2:289-294. Chung, S. and S. D. Kim. 2007. Escherichia coli can produce recombinant chitinase in the soil to control the pathogenesis by Fusarium oxysporum without colonization. J. Microbiol. Biotechnol. 17:474-480. Cohen-Kupiec, R. and I. Chet. 1998. The molecular biology of chitin digestion. Curr. Opin. Biotechnol. 9:270-277. Dahiya, N., R. Tewari, G. S. Hoondal. 2006. Biotechnological aspects of chitinolytic enzymes: a review. Appl. Microbiol. Biotechnol. 71:773-782. De Boer, W., P. J. A. Klein Gunnewiek, G. A. Kowalchuk, J. A. Van-Veen. 2001. Growth of chitinolytic dune soil beta-subclass Proteobacteria in response to invading fungal hyphae. Appl. Environ. Microbiol. 67:3358-3362. De la Vega, L. M., J. E. Barboza-Corona, M. G. Aguilar-Uscanga, M. Ramírez-Lepe. 2006. Purification and characterization of an exochitinase from Bacillus thuringiensis subsp. aizawai and its action against phytopathogenic fungi. Can. J. Microbiol. 52:651-657. Fogliano, V., A. Ballio, M. Gallo, S. Woo, F. Scala, M. Lorito. 2002. Pseudomonas lipodepsipeptides and fungal cell wall-degrading enzymes act synergistically in biological control. Mol. Plant-Microbe Interact. 15:323-333. Folders, J., J. Algra, M. S. Roelofs, L. C. Van Loon, J. Tommassen, W. Bitter. 2001. Characterization of Pseudomonas aeruginosa chitinase, a gradually secreted protein. J. Bacteriol. 183:7044-7052. Giambattista, R. D., F. Federici, M. Petruccioli, M. Fence. 2001. The chitinolytic activity of Penicillium janthinellum P9: purification, partial characteriza-

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tion and potential application. J. Appl. Microbiol. 91:498-505. Hong J. K. and B. K. Hwang. 2005. Functional characterization of PR-1 protein, ß-1,3-glucanase and chitinase genes during defense response to biotic and abiotic stresses in Capsicum annuum. Plant Pathol. J. 21:195-206. Hoster, F. J., E. Schmitz, R. Daniel. 2005. Enrichment of chitinolytic microorganisms: Isolation and characterization of a chitinase exhibiting antifungal activity against phytopathogenic fungi from novel Streptomyces strain. Appl. Microbiol. Biotechnol. 66:434-442. Jung, W. J., S. J. Jung, K. N. An, Y. L. Jin, R. D. Park, K. Y. Kim, B. K. Shon, T. H. Kim. 2002. Effect of chitinase-producing Paenibacillus illinoisensis KJA-424 on egg hatching of root-knot nematode (Meloidogyne incognita). J. Microbiol. Biotechnol. 12:865-871. Kuk, J. H., W. J. Jung, G. H. Jo, Y. C. Kim, K. Y. Kim, R. D. Park. 2005. Production of N-acetyl-b-D-glucosamine from chitin by Aeromonas sp. GJ-18 crude enzyme. Appl. Microbiol. Biotechnol. 68:384-389. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277:680-685. Lingappa, Y. and J. L. Lockwood. 1962. Chitin media for selectived culture of actinomycetes. Phytopathology. 52:317-323. Mathivanan, N., V. Kabilan, K. Murugesan. 1998. Purification, characterization, and antifungal activity of chitinase from Fusarium chlamydosporum, a mycoparasiteto groundnut rust, Puccinia arachidis. Can. J Microbiol. 44:646-651. Neiendam, N. M., and J. Sorensen. 1999. Chitinolytic activity of Pseudomonas fluorescens isolates from barley and sugarbeet rhizosphere. FEMS Microbiol. Ecol. 30:217-227. Thamthiankul, S., S. Suan-Ngay, S. Tantimavanich, W. Panbangred. 2001. Chitinase from Bacillus thuringiensis subsp. pakistani. Appl. Microbiol. Biotechnol. 56:395-401. Trudel, J. and A. Asselin. 1989. Detection of chitinase activity after polyacrylamide gel electrophoresis. Anal. Biochem. 178:362-366.

Van, N. N., Y. J. Kim, K. T. Oh, W. J. Jung, R. D. Park. 2008. Antifungal activity of chitinases from Trichoderma aureoviride DY-59 and Rhizopus microsporus VS-9. Curr. Microbiol. 56:28-32. Wang, S. L. and W. T. Chang. 1997. Purification and characterization of two bifunctional chitinase/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium. Appl. Environ. Microbiol. 63:380-386. Weisburg, W. G., S. M. Barns, D. J. Lane. 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173:697-703.

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