• Microbiology and Biotechnology Letters
• /
• v.28 no.3
• /
• pp.161-166
• /
• 2000

Antifungal Activity and Identification of an Actinomycetes Strain Isolated from Mummified Peaches. Lirn, Tae Heon*, Jung Mok Lee, Tae Hyun Chang, and Byeongjin Chal. *Research Institute of Plant Nutrient, Oaeyu Co, Inc. Kyongsan 712-820, Korea, 1 Department of Agricultural Bi%g'f Chungbul< NatJ"onal Univershy, Cheongju 367-763, Korea - An actinomycetes strain which produced chitinase, urease, and antifungal substances to MoniliniaJhtcticola was isolated from peaches mununified by Moniliniafructicola. The strain TH-04 was identified as Streptomyces sp. based on cultural and lTIOIphological characteristics, cell wall diaminopimelic acid, and sugar patterns ofwhole~cell extracts. Streptomyces sp. TH~04 showed antifungal activity to several fungi including Moniliniafructicola, Colletotrichum gloeosporioides, Magnaponhe grisea, Rhizoctonia solani, Phytophthora capsici, Altemaria kikuchiana, Fusarium solani, and Fusarium O),ysporum. The optimum cultural conditions for the production of antifungal substances were $20^{\circ}C$pH 7, and 7 days.

• The Plant Pathology Journal
• /
• v.28 no.4
• /
• pp.439-445
• /
• 2012

The chitinase producing Lysobacter enzymogenes C-3 has previously been shown to suppress plant pathogens in vitro and in the field, but little is known of the regulation of chitinase production, or its role in antimicrobial activity and biocontrol. In this study, we isolated and characterized chitinase-defective mutants by screening the transposon mutants of L. enzymogenes C-3. These mutations disrupted genes involved in diverse functions: glucose-galactose transpoter (gluP), disulfide bond formation protein B (dsbB), Clp protease (clp), and polyamine synthase (speD). The chitinase production of the SpeD mutant was restored by the addition of exogenous spermidine or spermine to the bacterial cultures. The speD and clp mutants lost in vitro antifungal activities against plant fungal pathogens. However, the gluP and dsbB mutants showed similar antifungal activities to that of the wild-type. The growth of the mutants in nutrient rich conditions containing chitin was similar with that of the wild-type. However, growth of the speD and gluP mutants was defective in chitin minimal medium, but was observed no growth retardation in the clp and dsbB mutant on chitin minimal medium. In this study, we identified the four genes might be involved and play different role in the production of extracellular chitinase and antifungal activity in L. enzymogenes C-3.

• The Plant Pathology Journal
• /
• v.25 no.4
• /
• pp.344-351
• /
• 2009

A biocontrol bacterium Bacillus licheniformis N1 grown in nutrient broth showed no chitinolytic activity, while its genome contains a gene which encodes a chitinase. The gene for chitinase from B. licheniformis N1 was amplified by PCR and the deduced amino acid sequence analysis revealed that the chitinase exhibited over 95% identity with chitinases from other B. licheniformis strains. Escherichia coli cells carrying the recombinant plasmid displayed chitinase activity as revealed by the formation of a clear zone on chitin containing media, indicating that the gene could be expressed in E. coli cells. Chitinase gene expression in B. licheniformis N1 was not detected by RT-PCR analysis. The protein was over-expressed in E. coli BL21 (DE3) as a glutathione S-transferase fusion protein. The protein could also be produced in B. subtilis 168 strain carrying the chitinase gene of N1 strain. The crude protein extract from E. coli BL21 carrying GST fusion protein or culture supernatant of B. subtilis carrying the chitinase gene exhibited enzyme activity by hydrolyzing chitin analogs, 4-methylumbelliferyl-$\beta$-D-N,N'-diacetylchitobioside and 4-methylumbelliferyl-$\beta$-D-N,N',N"-triacetylchitotrioside. These results indicated that even though the chitinase gene is not expressed in the N1 strain, the coding region is functional and encodes an active chitinase enzyme. Furthermore, B. subtilis 168 transformants expressing the chitinase gene exhibited antifungal activity against Fulvia fulva by suppressing spore germination. Our results suggest that the proper engineering of the expression of the indigenous chitinase gene, which will lead to its expression in the biocontrol strain B. licheniformis N1, may further enhance its biocontrol activity.

• Korean Journal of Environmental Agriculture
• /
• v.30 no.3
• /
• pp.346-356
• /
• 2011

BACKGROUND: Chemical fungicides not only may pollute the ecosystem but also can be environmentally hazardous, as the chemicals accumulate in soil. Biological control is a frequently-used environment-friendly alternative to chemical pesticides in phytopathogen management. However, the use of microbial products as fungicides has limitations. This study isolated and characterized a three-antifungal-enzyme (chitinase, cellulase, and ${\beta}$-1,3-glucanase)-producing bacterium, and examined the conditions required to optimize the production of the antifungal enzymes. METHOD AND RESULTS: The antifungal enzymes chitinase, cellulase, and ${\beta}$-1,3-glucanase were produced by bacteria isolated from an sawmill in Korea. Based on the 16S ribosomal DNA sequence analysis, the bacterial strain AM50 was identical to Streptomyces sp. And their antifungal activity was optimized when Streptomyces sp. AM50 was grown aerobically in a medium composed of 0.4% chitin, 0.4% starch, 0.2% ammonium sulfate, 0.11% $Na_2HPO_4$, 0.07% $KH_2PO_4$, 0.0001% $MgSO_4$, and 0.0001% $MnSO_4$ at $30^{\circ}C$. A culture broth of Streptomyces sp. AM50 showed antifungal activity towards the hyphae of plant pathogenic fungi, including hyphae swelling and lysis in P. capsici, factors that may contribute to its suppression of plant pathogenic fungi. CONCLUSION(S): This study demonstrated the multiantifungal enzyme production by Streptomyces sp. AM50 for the biological control of major plant pathogens. Further studies will investigate the synergistic effect, to the growth regulations by biogenic amines and antifungal enzyme gene promoter.

• Korean Journal Plant Pathology
• /
• v.10 no.4
• /
• pp.277-283
• /
• 1994

An antimicrobial chitinase was purified from grapefruit extract and its properties were characterized. The chitinase was purified with a single step chromatography on regenerated chitin affinity gel column. The molecular weight of the purified chitinase was 29 kDa. The grapefruit extract contained the chitinase protein more than 50% of its total soluble proteins measured by coomassie stained protein bands. When the purified chitinase was incubated with polymers of N-acetylglucosamine (NAG), such as mycelia of Fusarium oxysproum and swollen chitin, they were degraded to oligosaccharides, and the oligosaccharides were then further hydrolyzed by the same enzyme to monomer and dimer of NAG. This result suggests that the chitinase contained both endo- and exo- chitinase activities. The chitinase was stable to heat and pH treatment; its activity was not diminished by the heat treatment upto 7$0^{\circ}C$ for 1 hr, and it showed a pH stability in the range of pH 4.0 to 12.0.

• Journal of Microbiology and Biotechnology
• /
• v.7 no.2
• /
• pp.107-113
• /
• 1997

A strain producing a high amount of chitinase was isolated from soil, identified as Pseudomonas sp., and tentatively named Pseudomonas sp. YHS-A2. An extracellular chitinase of Pseudomonas sp. YHS-A2 was purified according to the procedure of ammonium sulfate saturation, affinity adsorption, Sephadex G-100 gel filtration and Phenyl-sepharose CL-4B hydrophobic interaction column chromatography. The molecular weight of the purified enzyme was estimated to be 55 kDa on SDS-PAGE was confirmed by active staining. Optimal pH and temperature of the enzyme are pH 7.0 and $50^{\circ}C$, respectively, and the enzyme is stable between pH 5.0 and 8.0 and below $50^{\circ}C$. The main products of colloidal chitin by the chitinase were N-acetyl-D-glucosamine and N,N'-diacetylchitobiose both of which were detected by HPLC analysis. The enzyme is supposed to be a random-type endochitinase which can degrade any position of ${\beta}$-l,4-linkages of chitin and chitooligosaccharides. The chitinase inhibited the growth of some phytopathogenic fungi, Fusarium oxysporum, Botrytis cineria, and Mucor rouxii and these antifungal effects were thought to be due to the characteristics of endochitinase.

• The Plant Pathology Journal
• /
• v.22 no.4
• /
• pp.323-328
• /
• 2006

Serratia marcescens W1, isolated from cucumber-cultivated soil in Suwon, Korea, evidenced profound antifungal activity and produced the extracellular hydrolytic enzymes, chitinase and protease. In order to isolate the antifungal genes from S. marcescens W1, a cosmid genomic library was constructed and expressed in Escherichia coli. Transformants exhibiting chitinase and protease expression were selected, as well as those transformants evidencing antifungal effects against the rice blast fungus, Magnaporthe grisea, and the cucumber leaf spot fungus, Cercospora citrullina. Cosmid clones expressing chitinase or protease exerted no inhibitory effects against the growth of fungal pathogens. However, two cosmid clones evidencing profound antifungal activities were selected for further characterization. An 8.2 kb HindIII fragment from these clones conditioned the expression of antagonistic activity, and harbored seven predicted complete open reading frames(ORFs) and two incomplete ORFs. The deduced amino acid sequences indicated that six ORFs were highly homologous with genes from S. marcescens generating pyrroloquinoline quinone(PQQ). Only subclones harboring the full set of pqq genes were shown to solubilize insoluble phosphate and inhibit fungal pathogen growth. The results of this study indicate that the functional expression of the pqq genes of S. marcescens W1 in E. coli may be involved in antifungal activity, via as-yet unknown mechanisms.

• Korean Journal of Microbiology
• /
• v.44 no.3
• /
• pp.258-263
• /
• 2008

Late blight, one of the most important disease in many agricultural crops, is caused by Phytophthora infestans. Fusarium wilt is a vascular disease of many plants caused by Fusarium oxysporum. Some bacteria isolated from rhizosphere were screened for their ability to inhibit the growth of F. oxysporum and P. infestans. Productions of siderophore, $\beta-1$,3-glucanase, hydrogen cyanide and chitinase by 4 isolated strains were examined. Among them, Bacillus sp. RFO41 most effectively inhibited the growth of F. oxysporum. The highest productions of siderophore and $\beta-l$,3-glucanase were shown in the culture of Bacillus sp. RFO41. Bacillus strain PS2 was most effective against P. infestans. PS2 showed the highest production of chitinase and hydrogen cyanide. A significant relationship was shown between the antagonistic effects of isolates against F. oxysporum and P. infestans and their production level of siderophore, $\beta-1$,3-glucanase, hydrogen cyanide, and chitinase.

• Microbiology and Biotechnology Letters
• /
• v.30 no.2
• /
• pp.135-141
• /
• 2002

This study was carried out to isolate antagonistic bacterium against Sclerotium cepivorum causing Allium fistulosum white rot. Total of 146 strains were isolated from A. fistulosum roots. The isolates were screened for antagonism to S. cepivorum and the isolated strain No. AL-1 was selected among these bacteria. It was identified as Serratia plymuthica based on morphological and physiological characteristics according to the Bergey's mannual of systematic bacteriology and 16S rDNA sequences methods. Serratia plymuthica AL-1 showed broad spectrum of antifungal activities against plant pathogenic fungi Alternaria altrata, Colletotrichum gleosporioids, Phoma sp., Rhizoctonia solani, Sclerotinia sclerotiorum, Stemphylium solani, Fusarium oxysporium niveum but not inhibited Didymella bryoniae. When S. plymuthica AL-1 cultivated in the TSB medium containing 1％ colloidal chitin, the high molecular fraction (>10 kDa) have chitinase activity (3.2 units/ml) and the low molecular fraction (<10 kDa) have not chitinase activity. Oppositely, after heat treatment (80℃ for 30 min) of the cultivation supernatant, the high molecular fractions have not antifungal activity but the low molecular fractions have antifungal activity.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.10
• /
• pp.1359-1366
• /
• 2012

A strain of Streptomyces cavourensis subsp. cavourensis (coded as SY224) antagonistic to Colletotrichum gloeosporioides infecting pepper plants was isolated. SY224 produced lytic enzymes such as chitinase, ${\beta}$-1,3-glucanase, lipase, and protease in respective assays. To examine for antifungal activity, the treatments amended with the nonsterilized supernatant resulted in the highest growth inhibition rate of about 92.9% and 87.4% at concentrations of 30% and 10%, respectively. However, the sterilized treatments (autoclaved or chloroform treated) gave a lowered but significant inhibitory effect of about 63.4% and 62.6% for the 10% supernatant concentration, and 75.2% and 74.8% for the of 30% supernatant concentration in the PDA agar medium, respectively, indicative of the role of a non-protein, heat stable compound on the overall effect. This antifungal compound, which inhibited spore germination and altered hyphal morphology, was extracted by EtOAc and purified by ODS, silica gel, Sephadex LH-20 column, and HPLC, where an active fraction was confirmed to be 2-furancarboxaldehyde by GS-CI MS techniques. These results suggested that SY224 had a high potential in the biocontrol of anthracnose in pepper, mainly due to a combined effect of lytic enzymes and a non-protein, heat-stable antifungal compound, 2-furancarboxaldehyde.