Sang-Dal Kim

Isolation and partial characterization of Bacillus subtilis ME488 for suppression of soilborne pathogens of cucumber and pepper

Environmentally friendly control measures are needed for suppression of soilborne pathogens of vegetable crops in the Republic of Korea. In vitro challenge assays were used to screen approximately 500 bacterial isolates from 20 Korean greenhouse soils for inhibition of diverse plant pathogens. One isolate, Bacillus subtilis ME488, suppressed the growth of 39 of 42 plant pathogens tested. Isolate ME488 also suppressed the disease caused by Fusarium oxysporum f. sp. cucumerinum on cucumber and Phytophthora capsici on pepper in pot assays. Polymerase chain reaction was used to screen isolate ME488 for genes involved in biosynthesis of 11 antibiotics produced by various isolates of B. subtilis. Amplicons of the expected sizes were detected for bacD and bacAB, ituC and ituD, and mrsA and mrsM involved in the biosynthesis of bacilysin, iturin, and mersacidin, respectively. The identity of these genes was confirmed by DNA sequence analysis of the amplicons. Bacilysin and iturin were detected in culture filtrates from isolate ME488 by gas chromatography coupled with mass spectroscopy and by thin layer chromatography, respectively. Detection of mersacidin in ME488 culture filtrates was not attempted. Experiments reported here indicate that B. subtilis ME488 has potential for biological control of pathogens of cucumber and pepper possibly due to the production of antibiotics.

DNA extraction conditions fromPorphyra perforata using LiCl

A rapid and economical method of DNA extraction from a red seaweedPorphyra perforata J. Agardh has been developed by the use of lithium chloride. This paper describes the optimization of extraction conditions. Heat treatment of tissues in a solution (0.8 M LiCl, 0.6% Sarkosyl, 10 mM EDTA, 0.2% PVPP, 5% ß-mercaptoethanol, pH 9.0) at 55 °C for 10 min extracts DNA that is of sufficient quality to be used as a template for PCR amplification. Total DNA yield was approximately 30 to 50μg g−1 t of partially dried tissue. Total RNA yield was approximately 400μg g−1 of partially dried tissue. Carbohydrate was contained as approximately 40 to 90 mM (expressed as glucose equivalents) from 1 g tissue, and protein contamination calculated as the O.D. 260/280 ratio was in the range of 1.4 to 1.7. The DNA was characterized by high molecular weight larger than 50 kb.

Isolation of Endophytic Fungi Capable of Plant Growth Promotion from Monocots Inhabited in the Coastal Sand Dunes of Korea

Endophytic fungi predominantly inhabit grasses, and produce a variety of beneficial metabolites for plant growth, as well as help their hosts against pathogens and herbivores. Current study was focused on plant growth promoting activity of endophytic fungi inhabited in the roots of sand dune grasses. We collected 49 fungal isolates from the roots of four most common sand dune grasses and screened them for their growth promoting capacity. Results showed that 37 fungal isolates (75.5%) promoted plant height and shoot length of waito-c rice, 11 fungal isolates (22.5%) suppressed it, while 1 fungus (2%) showed no effect on the growth attributes. The fungal strain Gibberella fujikuroi, along with distilled water and Czapek broth medium, were taken as control for this experiment. It was concluded that a major proportion of endophytic fungi inhabited in the sand dune plants produce metabolites, and thus help in growth and development of the host plant.

Isolation of auxin- and 1-aminocyclopropane-1-carboxylic acid deaminase-producing bacterium and its effect on pepper growth under saline stress

Plant tissues produce ethylene under the environmental stresses such as drought, salinity, and heavy metals. Ethylene concentration can be reduced by 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase produced by plant growth-promoting rhizobacterium (PGPR), which cleaves the ethylene precursor ACC. The present study focused on alleviation of environmental stress by selected PGPR, which could suppress fungal plant disease. These PGPRs were capable of utilizing ACC as sole source of nitrogen and also produced auxin. Seed germination of red pepper was reduced with increasing salt concentration, and approximately 98.2% of seeds germinated in the absence of salt, whereas only 36.2% seeds germinated in the presence of 175 mM NaCl. Seed germination was also decreased by 62.1 and 19.9% in the presence of 120mM NaCl and 120mM NaCl +ACC deaminase-producing PGPR Pseudomonas fluorescens 2112, respectively, compared to uninoculated control. The effect of salinity stress with different salt concentration on pepper plants and their alleviation with PGPR was evaluated. Non-inoculated pepper plants died after 5 week when grown in the presence of high salt (120 mM NaCl), whereas 80% of pepper plants inoculated with P. fluorescens 2112 survived under the high salt stress. Salt stress also decreased the fresh and dry weights of pepper grown, compared to the negative control, whereas pepper plants inoculated with P. fluorescens 2112 retained the biomass similar to control plants. These results indicate that ACC deaminase and auxin producing P. fluorescens 2112 is a multi-functional PGPR that can promote the growth and development of pepper plants by alleviating the high-salt stress.

Cloning and sequencing of a novel glutaryl acylase β-subunit gene ofPseudomonas cepacia BY21 from bioinformatics

Pseudomonas cepacia BY21 was found to produce glutaryl acylase that is capable of deacylating glutaryl-7-aminocephalosporanic acid (glutaryl-7-ACA) to 7-aminocephalosporanic acid (7-ACA), which is a starting material for semi-synthetic cephalosporin antibiotics. Amino acids of the reported glutaryl acylases from variousPseudomonas sp. strains show a high similarity (>93% identity). Thus, with the known nucleotide sequences ofPseudomonas glutaryl acylases in GenBank, PCR primers were designed to clone a glutaryl acylase gene fromP. cepacia BY21. The unknown β-subunit gene of glutaryl acylase from chromosomal DNA ofP. cepacia BY21 was cloned successfully by PCR. The β-subunit amino acids ofP. cepacia BY21 acylase (GenBank accession number AY948547) were similar to those ofPseudomonas diminuta KAC-1 acylase except that Asn408 ofP. diminuta KAC-1 acylase was changed to Leu408.

Root Colonization and Quorum Sensing of the Antagonistic Bacterium Pseudomonas fluorescens 2112 involved in the Red-pepper Rhizosphere

Biofilm formation of multifunctional plant growth promoting rhizobacterium (PGPR), Pseudomonas fluorescens 2112 is necessary for P. fluorescens 2112 to have a positive impact on the rhizosphere of red-pepper. This study investigated whether signal molecules of the quorum sensing AHLs are produced in order to confirm biofilm formative ability. Through the use of Petri dish bioassays a blue circle formed evidence of AHLs. It was confirmed that P. fluorescens 2112 produced six-carbon-chain-long AHLs by TLC bioassay. The bacterial density of P. fluorescens 2112 on the top and bottom of pepper plant roots was estimated as and CFU/g root, respectively. P. fluorescens 2112 exist more with high-density of CFU/g soil at a depth of 1 cm but at a low-density of CFU/g soil at a depth of 5 cm, from the surface of rhizosphere soil. In addition, biofilm formation of P. fluorescens 2112 on the epidermises and the tips of the red-pepper roots were confirmed visually by SEM. Thus, the production of AHLs by P. fluorescens 2112 brings about quorum sensing signaling and the formation of biofilm on the roots which has a positive effect on economically important crops such as red-pepper by additionally producing a variety of antifungal substances and auxin.

Effects on the Soil Microbial Diversity and Growth of Red Pepper by Treated Microbial Agent in the Red Pepper Field

We investigated the effects on soil microbial diversity and the growth promotion of red pepper resulting from inoculation with a microbial agent composed of Bacillus subtilis AH18, B. licheniformis K11 and Pseudomonas fluorescens 2112 in a red pepper farming field. Photosynthetic bacteria, Trichoderma spp., Azotobacter spp., Actinomycetes, nitrate oxidizing bacteria, nitrite oxidizing bacteria, nitrogen fixing bacteria, denitrifying bacteria, phosphate solubilizing bacteria, cellulase producing bacteria, and urease producing bacteria are all indicator microbes of healthy soil microbial diversity. The microbial diversity of the consortium microbial agent treated soil was seen to be 1.1 to 14 times greater than soils where other commercial agent treatments were used, the latter being the commercial agent AC-1, and chemical fertilizer. The yield of red pepper in the field with the treated consortium microbial agent was increased by more than 15% when compared to the other treatments. Overall, the microbial diversity of the red pepper farming field soil was improved by the consortium microbial agent, and the promotion of growth and subsequent yield of red pepper was higher than soils where the other treatments were utilized.

Selection and Identification of Phytohormones and Antifungal Substances Simultaneously Producing Plant Growth Promoting Rhizobacteria from Microbial Agent Treated Red-pepper Fields

In this study, a total of more than 1,000 bacteria, including 739 species of aerobic bacteria, 80 species of urease producing bacteria and 303 species of photosynthetic bacteria, were isolated from red-pepper field soils located in the Gyeongsan Province of the Republic of Korea. Amongst these, 158 species of aerobic bacteria, 70 species of urease producing bacteria and 228 species of photosynthetic bacteria were found to be auxin producing soil bacteria through quantification analysis with the Salkowski test. The latter groupings were then tested for antifungal activities to -Glucanase and siderophore using CMC congo red agar and CAS blue agar media. In addition, the selected strains were examined for antifungal activity against various phytopathogenic fungi on PDN agar media. Six strains; BCB14, BCB17, C10, HA46, HA143, and HJ5, were noted for their ability to both produce auxin and act as antifungal substances. 16S rDNA sequence comparison analyses of these six strains identified them as Bacillus subtilis BCB14, B. methylotrophicus BCB17, B. methylotrophicus C10, B. sonorensis HA46, B. subtilis HA143, and B. safensis HJ5.