Kil Yong Kim

GacS-Dependent Production of 2R, 3R-Butanediol by Pseudomonas chlororaphis O6 Is a Major Determinant for Eliciting Systemic Resistance Against Erwinia carotovora but not Against Pseudomonas syringae pv. tabaci in Tobacco

Root colonization by a plant-beneficial rhizobacterium, Pseudomonas chlororaphis O6, induces disease resistance in tobacco against leaf pathogens Erwinia carotovora subsp. carotovora SCC1, causing soft-rot, and Pseudomonas syringae pv. tabaci, causing wildfire. In order to identify the bacterial determinants involved in induced systemic resistance against plant diseases, extracellular components produced by the bacterium were fractionated and purified. Factors in the culture filtrate inducing systemic resistance were retained in the aqueous fraction rather than being partitioned into ethyl acetate. Fractionation on high-performance liquid chromatography followed by nuclear magnetic resonance mass spectrometry analysis identified the active compound as 2R, 3R-butanediol. 2R, 3R butanediol induced systemic resistance in tobacco to E. carotovora subsp. carotovora SCC1, but not to P. syringae pv. tabaci. Treatment of tobacco with the volatile 2R, 3R-butanediol enhanced aerial growth, a phenomenon also seen in plants colonized by P. chlororaphis O6. The isomeric form of the butanediol was important because 2S, 3S-butandiol did not affect the plant. The global sensor kinase, GacS, of P. chlororaphis O6 was a key regulator for induced systemic resistance against E. carotovora through regulation of 2R, 3R-butanediol production. This is the first report of the production of these assumed fermentation products by a pseudomonad and the role of the sensor kinase GacS in production of 2R, 3R-butanediol.

Extracellular proteins involved in soybean cultivar-specific nodulation are associated with pilus-like surface appendages and exported by a type III protein secretion system in Sinorhizobium fredii USDA257

Several gram-negative plant and animal pathogenic bacteria have evolved a type III secretion system (TTSS) to deliver effector proteins directly into the host cell cytosol. Sinorhizobium fredii USDA257, a symbiont of soybean and many other legumes, secretes proteins called Nops (nodulation outer proteins) into the extracellular environment upon flavonoid induction. Mutation analysis and the nucleotide sequence of a 31.2-kb symbiosis (sym) plasmid DNA region of USDA257 revealed the existence of a TTSS locus in this symbiotic bacterium. This locus includes rhc (rhizobia conserved) genes that encode components of a TTSS and proteins that are secreted into the environment (Nops). The genomic organization of the TTSS locus of USDA257 is remarkably similar to that of another broad-host range symbiont, Rhizobium sp. strain NGR234. Flavonoids that activate the transcription of the nod genes of USDA257 also stimulate the production of novel filamentous appendages known as pili. Electron microscope examination of isolated pili reveals needle-like filaments of 6 to 8 nm in diameter. The production of the pili is dependent on a functional nodD1 and the presence of a nod gene-inducing compound. Mutations in several of the TTSS genes negate the ability of USDA257 to elaborate pili. Western blot analysis using antibodies raised against purified NopX, Nop38, and Nop7 reveals that these proteins were associated with the pili. Mutations in rhcN, rhcJ, rhcC, and ttsI alter the ability of USDA257 to form nodules on Glycine max and Macroptilium atropurpureum.

2-Ketogluconic Acid Production and Phosphate Solubilization by Enterobacter intermedium

Enterobacter intermedium, isolated from grass rhizosphere, exhibited a strong ability to solubilize insoluble phosphate. This bacterium oxidized glucose to gluconic acid and sequentially to 2-ketogluconic acid (2-KGA), which was identified using HPLC and GC-MS. The ability of E. intermedium to solubilize phosphate and produce 2-KGA produce in broth medium containing different components was monitored with air and without air supply. With an air supply, the production of 2-KGA markedly increased to about 110 g/l at day 10 in media containing 0.2 M gluconic acid, while it was about 65 g/l without gluconic acid addition. With an air supply, the concentration of soluble phosphate significantly decreased to 200–250 mg/l in media containing 1% CaCO3, whereas it was about 1000 mg/l without CaCO3 addition. Without an air supply, the concentration of 2-KGA and phosphate were negligible throughout the culture period. RID=”” ID=”” Correspondence to: K.Y. Kim; email: kimkil@chonnam.ac.kr

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.

Cloning and expression of pyrroloquinoline quinone (PQQ) genes from a phosphate-solubilizing bacterium Enterobacter intermedium.

A grass rhizosphere bacterium, Enterobacter intermedium (60-2G), has a strong ability to solubilize insoluble phosphate. Certain phosphate-solubilizing bacteria secrete gluconic acid for this process. The gluconic acid is produced by direct extracellular oxidation of glucose by a glucose dehydrogenase equipped with pyrroloquinoline quinone (PQQ) as a cofactor. A pqq gene cluster producing PQQ was detected in E. intermedium and this sequence conferred phosphate-solubilizing activity to Escherichia coli DH5α. The 6,783-bp pqq sequence had six open reading frames (pqqA, B, C, D, E, and F) and showed 50–95% homology to pqq genes of other bacteria. E. coli DH5α expressing the E. intermedium pqq genes solubilized phosphate from hydroxyapatite after a pH drop to pH 4.0, which paralleled in time the secretion of gluconic acid. We speculate that production of PQQ in E. coli DH5α expressing the pqq cluster activates an endogenous glucose dehydrogenase to permit gluconic acid secretion that solubilizes the insoluble phosphate.

Biological control of damping-off caused by Rhizoctonia solani using chitinase-producing Paenibacillus illinoisensis KJA-424

Abstract A bacterium having strong chitinolytic activity was isolated from a coastal soil in Korea and identified as Paenibacillus illinoisensis KJA-424 on the basis of the nucleotide sequence of a 16S rRNA gene. By activity staining after SDS–PAGE, three major chitinase bands with chitinolytic activity, approximate molecular weight of 63, 54 and 38 kDa were detected. On co-culture Rhizoctonia solani with KJA-424, abnormal swelling and deformation of R. solani hyphae were observed, where the release of N -acetyl- d -glucosamine was detected. The bacterium suppressed the symptom of damping-off cucumber seedlings caused by R. solani , in greenhouse trial.

Effect of the different timing of AMF inoculation on plant growth and flower quality of chrysanthemum

Abstract Plant growth and flower quality of an ornamental plant (Chrysanthemum morifolium Ramat) var. Baekgwang in response to the different timing of arbuscular mycorrhizal fungi (AMF) inoculation were examined. To evaluate the effects of AMF inoculation timing on growth of chrysanthemum cuttings, AMF was directly inoculated to cutting (AMFD), or inoculated at transplanting stage (AMFA). Control plants did not receive AMF inoculation (CON). A significant difference in plant growth was observed in AMF treatment compared to non-inoculated treatment at transplanting stage. Rooting rate in AMF treatment was 99% whereas it was 76% in non-mycorrhizal inoculation. The colonization rate was 3.9% in AMF treatment, while it was 0% in non-AMF treatment. Tap root length and number of lateral roots in AMF treatment were twice than those of non-AMF treatment. Inoculation of AMF significantly increased shoot and root growth at transplanting stage. At 7 weeks after transplanting, chrysanthemum plants in AMFD and AMFA treatments had 46.42 and 34.24% colonization rate, respectively. Low colonization rate (6.08%) was observed in CON, possibly due to contamination. Plant height, leaf area, root length, and fresh and dry weight of shoots, stems and roots in AMF inoculation treatments (AMFD and AMFA) were significantly higher than control plants. AMF inoculation significantly shortened flowering time compared to non-AMF plants. Chrysanthemum in AMFD, AMFA and CON treatments flowered at 98, 104 and 112 days after transplanting, respectively. Fresh weight, width and height of flowers in AMFD and AMFA treatments were generally higher than those in CON. However, a significant increase in fresh weight, width and height of flowers was found in AMFD treatment. A significant increase of macronutrient uptake in leaves was observed for AMFD treatment, compared with CON. Mn uptake in AMFD and AMFA was more than double than that in CON. In roots, macro and micronutrient concentrations were generally higher in AMFD.

Extraction of chitin from red crab shell waste by cofermentation with Lactobacillus paracasei subsp. tolerans KCTC-3074 and Serratia marcescens FS-3

For one-step extraction of chitin from red crab shell waste, cofermentation with Lactobacillus paracasei subsp. tolerans KCTC-3074, a lactic-acid-producing bacterium, and Serratia marcescens FS-3, a protease-producing bacterium, was conducted. Fermentation with single strain (L. 3074 or FS-3) was also conducted. At day 7, the pH in L. 3074, FS-3, and L. 3074+FS-3 (1:1) treatment decreased from 6.90 to 3.30, 5.88, and 3.48, respectively. Ash content in the residue after fermentation treatment of crab shells in L. 3074 and L. 3074+FS-3 (1:1) treatment drastically decreased from 41.2% to 3.19 and 1.15%, respectively. In L. 3074+FS-3 (1:1) cofermentation, the level of demineralization was the highest value of 97.2%, but the level of deproteinization in the cofermentation was 52.6% at day 7. Protein content in the treatment of FS-3 alone reduced from 22.4 to 3.62%. These results indicate that cofermentation of the shells using the two strains is efficient and applicable for the one-step extraction of crude chitin from red crab shell waste.

Multiple determinants influence root colonization and induction of induced systemic resistance by Pseudomonas chlororaphis O6

SUMMARY Colonization of the roots of tobacco by Pseudomonas chlororaphis O6 induces systemic resistance to the soft-rot pathogen, Erwinia carotovora ssp. carotovara SCC1. A screen of the transposon mutants of P. chlororaphis O6 showed mutants with about a fivefold reduction in ability to induce systemic resistance to the soft-rot disease. These mutations disrupted genes involved in diverse functions: a methyl-accepting chemotaxis protein, biosynthesis of purines, phospholipase C, transport of branched-chain amino acids and an ABC transporter. Additional mutations were detected in the intergenic spacer regions between genes encoding a GGDEF protein and fumarate dehydratase, and in genes of unknown function. The mutants in the ABC transporters did not display reduced root colonization. However, the other mutants had up to 100-fold reduced colonization levels. Generally the production of metabolites important for interactions in the rhizosphere, phenazines and siderophores, was not altered by the mutations. A reduced induction of systemic resistance by a purine biosynthesis mutant with a disrupted purM gene correlated with poor growth rate, lesser production of phenazines and siderophore and low levels of root colonization. These studies showed that multiple determinants are involved in the induction of systemic resistance, with there being a requirement for strong root colonization.

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.