Seur Kee Park

An effective biocontrol bioformulation against Phytophthora blight of pepper using growth mixtures of combined chitinolytic bacteria under different field conditions

Phytophthora blight of pepper caused by Phytophthora capsici has devastating consequences when combined with other pathogens, including Rhizoctonia solani, Fusarium oxysporum, and Fusarium solani. In order to develop a field-effective biocontrol strategy against Phytophthora blight of pepper, three chitinolytic bacteria, Serratiaplymuthica strain C-1, strongly antagonistic to P. capsici, Chromobacterium sp. strain C-61, strongly antagonistic to R. solani, and Lysobacter enzymogenes strain C-3, antagonistic to R. solani and Fusarium spp., were selected. In pot studies, application of cultures combining the three bacterial strains effectively suppressed Phytophthora blight more than application of any single bacterial strain. Bioformulations developed from growth of the strains in a simple medium containing chitin under large batch conditions resulted in effective control in field applications. Efficacy of the bioformulated product depended on both the dose and timing of application. Although the undiluted product suppressed Phytophthora blight under all field conditions, a 10-fold diluted product was effective in solar-sterilized greenhouses and in fields with crop rotation. These results suggest that the developed product could be a new effective system to control Phytophthora blight disease in pepper.

Influence of the Transposition of the Thermostabilizing Domain of Clostridium thermocellum Xylanase (XynX) on Xylan Binding and Thermostabilization

ABSTRACT A xylanase gene, xynX, of Clostridium thermocellum had one thermostabilizing domain (TSD) between the signal peptide sequence and the catalytic domain (CD). The TSD of a truncated xylanase gene, xynX′TSD-CD, was transpositioned from the N terminus to the C terminus of the CD by overlapping PCRs, and a modified product, xynX′CD-TSD, was constructed. XynX′TSD-CD had a higher optimum temperature (70°C versus 65°C) and was more thermostable (residual activity of 68% versus 46% after a 20-min preincubation at 70°C) than the one without the TSD, XynX′CD. However, the domain-transpositioned enzyme, XynX′CD-TSD, showed a lower optimum temperature (30°C) and thermostability (20%) than XynX′CD. Both XynX′TSD-CD and XynX′CD-TSD showed significantly higher binding capacity toward xylan than XynX′CD, and the domain transposition did not cause any change in the binding ability. XynX′TSD-CD and XynX′CD-TSD also showed considerable binding to lichenan but not to carboxymethyl cellulose and laminarin. XynX′TSD-CD and XynX′CD-TSD had higher activities for insoluble xylan than XynX′CD, while XynX′CD was more active against soluble xylan than XynX′TSD-CD and XynX′CD-TSD. These results indicate that the TSD of XynX has dual functions, xylan binding and thermostabilization, and the domain should also be classified as a xylan-binding domain (XBD). The binding capacity of the XBD was not affected by domain transpositioning within the gene.

Inactivation of pqq genes of Enterobacter intermedium 60‐2G reduces antifungal activity and induction of systemic resistance

Enterobacter intermedium 60-2G, a phosphate solubilizing bacterium, has the ability to induce systemic resistance in plants against soft rot pathogen Erwinia carotovora. Glucose dehydrogenase, an enzyme that utilizes pyrroloquinoline quinone (PQQ) as a cofactor, is required for the synthesis of gluconic acid by E. intermedium 60-2G. Here, we report that the pqqA and pqqB genes are required for phosphate solubilization and induced systemic resistance against a soft rot pathogen in tobacco. Mutations in either the pqqA or pqqB gene abolished the production of 2-ketogluconic acid and eliminated the ability of E. intermedium to solubilize hydroxyapatite. Addition of gluconic acid to the growth media restored the ability of the pqqA mutant to produce 2-ketogluconic acid. Interestingly, both pqqA and pqqB mutants of E. intermedium lost their ability to inhibit the growth of the rice pathogen Magnaporthe grisea KI-409. Additionally, induced systemic resistance against the soft rot pathogen was attenuated in the pqq mutants. These functions were restored by complementation with the wild-type pqq gene cluster. Our findings suggest that PQQ plays an important function in beneficial traits including phosphate solubilization, antifungal activity, and induced systemic resistance of E. intermedium, possibly by acting as a cofactor for several enzymes including glucose dehydrogenase.

Development of effective environmentally-friendly approaches to control Alternaria blight and anthracnose diseases of Korean ginseng

In ginseng fields, Alternaria blight, caused by Alternaria panax, and anthracnose, caused by Colletotrichum gloeosporoides, have become serious problems in Korea, and control of these diseases relies mainly on intensive applications of fungicides. In an effort to develop an effective environmentally-friendly control system, we utilized preventative control approaches including rain shelter shading plates and the removal of dead plants, as well as a curative control approach, such as the application of microbial agents. In the presence of rain shelter shading plates, the occurrence of Alternaria blight and anthracnose decreased significantly compared to that seen with polyethylene shading nets. In addition, the eradication of dead ginseng plants, which harboured abundant spores of the pathogens, significantly reduced the incidence of both diseases. In fields with rain shelter shading plates and in which dead plants were eradicated, four applications of a bioformulated product containing chitinolytic bacterial strains in a simple medium containing chitin provided control similar in effect to that observed with the application of the fungicide under low disease pressure. The efficacy of the bioformulated product was decreased slightly under severe disease pressure. These findings indicate that integration of the three disease management measures might constitute a new effective and environmentally-friendly system for the control of Alternaria blight and anthracnose in Korean ginseng fields.

Draft Genome Sequence of the Biocontrol Bacterium Chromobacterium sp. Strain C-61

Chromobacterium sp. strain C-61 is a plant-associated bacterium with proven capacities to suppress plant diseases. Here, we report the draft genome sequence and automatic annotation of strain C-61. A comparison of this sequence to the sequenced genome of Chromobacterium violaceum ATCC 12472 indicates the novelty of C-61 and a subset of gene functions that may be related to its biocontrol activities.

Insight Into Genes Involved in the Production of Extracellular Chitinase in a Biocontrol Bacterium Lysobacter enzymogenes C-3

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.

Characterization of a Homogentisate Dioxygenase Mutant in Pseudomonas chlororaphis O6

Transposon mutagenesis of Pseudomonas chlororaphis O6 was performed with the transposon Tn5 to investigate genes involved in production of secondary metabolites. A mutant, termed Org, produced intense dark-brown pigmentation on rich medium. The Tn5-flanking sequence of the Org mutant showed high homology with the hmgA gene encoding the enzyme homogentisate dioxygenase, involved in the degradation of aromatic amino acids such as tyrosine. Growth of the hmgA mutant on L-tyrosine as sole carbon and energy sources was impaired. Growth on L-tyrosine was restored and production of the brown melanin pigment was eliminated when the mutant was complemented with the wild-type hmgA gene. The change in aromatic amino acids metabolism caused by the deletion of the hmgA gene function did not impair production of phenazines and biological traits connected to these secondary compounds: inhibition of fungal growth and inhibition of barley seed germination.

Purification and Characterization of a Major Extracellular Chitinase from a Biocontrol Bacterium, Paenibacillus elgii HOA73

Chitinase-producing Paenibacillus elgii strain HOA73 has been used to control plant diseases. However, the antimicrobial activity of its extracellular chitinase has not been fully elucidated. The major extracellular chitinase gene (PeChi68) from strain HOA73 was cloned and expressed in Escherichia coli in this study. This gene had an open reading frame of 2,028 bp, encoding a protein of 675 amino acid residues containing a secretion signal peptide, a chitin-binding domain, two fibronectin type III domains, and a catalytic hydrolase domain. The chitinase (PeChi68) purified from recombinant E. coli exhibited a molecular mass of approximately 68 kDa on SDS-PAGE. Biochemical analysis indicated that optimum temperature for the actitvity of purified chitinase was 50ºC. However, it was inactivated with time when it was incubated at 40ºC and 50ºC. Its optimum activity was found at pH 7, although its activity was stable when incubated between pH 3 and pH 11. Heavy metals inhibited this chitinase. This purified chitinase completely inhibited spore germination of two Cladosporium isolates and partially inhibited germination of Botrytis cinerea spores. However, it had no effect on the spores of a Colletotricum isolate. These results indicate that the extracellular chitinase produced by P. elgii HOA73 might have function in limiting spore germination of certain fungal pathogens.

Draft Genome Sequence of a Chitinase-producing Biocontrol Bacterium Serratia sp. C-1

【The chitinase-producing bacterial strain C-1 is one of the key chitinase-producing biocontrol agents used for effective bioformulations for biological control. These bioformulations are mixed cultures of various chitinolytic bacteria. However, the precise identification, biocontrol activity, and the underlying mechanisms of the strain C-1 have not been investigated so far. Therefore, we evaluated in planta biocontrol efficacies of C-1 and determined the draft genome sequence of the strain in this study. The bacterial C-1 strain was identified as a novel Serratia sp. by a phylogenic analysis of its 16S rRNA sequence. The Serratia sp. C-1 bacterial cultures showed strong in planta biocontrol efficacies against some major phytopathogenic fungal diseases. The draft genome sequence of Serratia sp. C-1 indicated that the C-1 strain is a novel strain harboring a subset of genes that may be involved in its biocontrol activities.】