• Journal of Microbiology and Biotechnology
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• v.30 no.5
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• pp.717-725
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• 2020

The use of plant growth-promoting rhizobacteria is economically viable and environmentally safe for mitigating various plant stresses. Abiotic stresses such as flood and drought are a serious threat to modern agriculture. In the present study, the indole-3-acetic acid-producing rhizobacterium R. sphaeroides KE149 was selected, and its effects on the growth of adzuki bean plants under flood stress (FS) and drought stress (DS) were investigated. IAA quantification of bacterial pure culture revealed that KE149 produced a significant amount of IAA. Moreover, KE149 inoculation notably decreased stress-responsive endogenous abscisic acid and jasmonic acid and increased salicylic acid in plants under DS and FS. KE149 inoculation also increased proline under DS and methionine under FS. In addition, KE149 inoculation significantly increased the levels of calcium (Ca), magnesium (Mg), and potassium (K) while lowering the sodium (Na) content in the plant shoot under stress. KE149-treated plants had markedly greater root length, shoot length, stem diameter, biomass, and higher chlorophyll content under both normal and stressed conditions. These results suggest that KE149 could be an efficient biofertilizer for mitigating water stress.

• Journal of Microbiology and Biotechnology
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• v.30 no.1
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• pp.118-126
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• 2020

Silicon and phosphorus are elements that are beneficial for plant growth. Despite the abundant availability of silicate and phosphate in the Earth's crust, crop nutritional requirements for silicon and phosphorus are normally met through the application of fertilizer. However, fertilizers are one of the major causes of heavy metal pollution. In our study, we aimed to assess silicate and phosphate solubilization by the bacteria Enterobacter ludwigii GAK2, in the presence and absence of phosphate [Ca₃(PO₄)₂] or silicate (Mg₂O₈Si₃), to counteract cadmium stress in rice (Oryza sativa L). Our results showed that the GAK2-treated rice plants, grown in soil amended with phosphate [Ca₃(PO₄)₂] or silicate (Mg₂O₈Si₃), had significantly reduced cadmium content, and enhanced plant growth promoting characteristics including fresh shoot and root weight, plant height, and chlorophyll content. These plants showed significant downregulation of the cadmium transporter gene, OsHMA2, and upregulation of the silicon carrier gene, OsLsi1. Moreover, jasmonic acid levels were significantly reduced in the GAK2-inoculated plants, and this was further supported by the downregulation of the jasmonic acid related gene, OsJAZ1. These results indicate that Enterobacter ludwigii GAK2 can be used as a silicon and phosphorus bio-fertilizer, which solubilizes insoluble silicate and phosphate, and mitigates heavy metal toxicity in crops.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2010.05a
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• pp.17-17
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• 2010

Ethylene, known as a stress hormone regulate wide developmental processes including germination, root hair initiation, root and shoot primordial formation and elongation, leaf and flower senescence and abscission, fruit ripening. The acceleration of ethylene biosynthesis in plant associated with environmental and biological stresses. 1-Aminocycloprophane-1-carboxlyate deaminase(ACCD) is an enzyme that cleaves ACC into and ammonia, a precursor of the plant hormone ethylene. Plant growth-promoting rhizobacteria (PGPR) having ACCD can decrease endogenous ACC level of tissue, resulting in reduced production of ethylene in plants. ACC deaminse was a key enzyme for protect stressed plants from injurious effects of ethylene. ACCD gene was encoded from Pseudomonas flourescens, PGPR and was cloned in Escherichia coli. We expressed the recombinant ACCD(rACCD) containing 357 amino acids with molecular weight 39 kDa that revealed by SDS-PAGE and western blot. The rACCD was purified by Ni-NTA purification system. The active form of rACCD having enzyme activity converted ACC to a-ketobutyrate. The optimal pH for ACC deaminase activity was pH 8.5, but no activity below pH 7.0 and a less severe tapering activity at base condition resulting in loss of activity at over pH 11. The optimal temperature of the enzyme was $30^{\circ}$ and a slightly less severe tapering activity at 15 - 30$^{\circ}$, but no activity over $35^{\circ}$. P. flourescens ACC deaminase has a highly conserved residue that plays in allowing substrate accessibility to the active sites. The enzymatic properties of this rACCD will provide an important reference for analysis of newly isolated ACCD and identification of newly isolated PGPR containing ACCD.

• Journal of Microbiology and Biotechnology
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• v.21 no.4
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• pp.379-386
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• 2011

The objective of this work was to investigate the ability of the plant growth-promoting rhizobacterium Pseudomonas aureofaciens 63-28 to induce plant defense systems, including defense-related enzyme levels and expression of defense-related isoenzymes, and isoflavone production, leading to improved resistance to the phytopathogen Rhizoctonia solani AG-4 in soybean seedlings. Seven-day-old soybean seedlings were inoculated with P. aureofaciens 63-28, R. solani AG-4, or P. aureofaciens 63-28 plus R. solani AG-4 (P+R), or not inoculated (control). After 7 days of incubation, roots treated with R. solani AG-4 had obvious damping-off symptoms, but P+R-treated soybean plants had less disease development, indicating suppression of R. solani AG-4 in soybean seedlings. Superoxide dismutase (SOD) and catalase (CAT) activities of R. solani AG-4-treated roots increased by 24.6% and 54.0%, respectively, compared with control roots. Ascorbate peroxidase (APX) and phenylalanine ammonia lyase (PAL) activities of R. solani AG-4-treated roots were increased by 75.1% and 23.6%, respectively. Polyphenol oxidase (PPO) activity in soybean roots challenged with P. aureofaciens 63-28 and P+R increased by 25.0% and 11.6%, respectively. Mn-SOD (S1 band on gel) and Fe-SOD (S2) were strongly induced in P+R-treated roots, whereas one CAT (C1) and one APX (A3) were strongly induced in R. solani AG-4- treated roots. The total isoflavone concentration in P+Rtreated shoots was 27.2% greater than the control treatment. The isoflavone yield of R. solani AG-4-treated shoots was 60.9% less than the control.

• Journal of Microbiology and Biotechnology
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• v.24 no.1
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• pp.106-112
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• 2014

Very few plant growth-promoting rhizobacteria (PGPR) are known to produce gibberellins (GAs). The current study aimed to isolate a phytohormone-producing PGP rhizobacterium from soil and assess its potential to enhance plant growth. The newly isolated bacterium was identified as Leifsonia soli sp. SE134 on the basis of partial 16S ribosomal RNA gene sequence. Application of L. soli culture filtrate significantly increased the biomass, hypocotyl, and root lengths of cucumber seeds as compared with non-inoculated sole medium and distilled water treated controls. Furthermore, the PGPR culture was applied to the GA-deficient mutant rice cultivar Waito-C. Treatment with L. soli SE134 significantly increased the growth of Waito-C rice seedlings as compared with controls. Upon chromatographic analysis of L. soli culture, we isolated, detected and quantified different GAs; namely, $GA_1$ ($0.61{\pm}0.15$), $GA_4$ ($1.58{\pm}0.26$), $GA_7$ ($0.54{\pm}0.18$), $GA_8$ ($0.98{\pm}0.15$), $GA_9$ ($0.45{\pm}0.17$), $GA_{12}$ ($0.64{\pm}0.21$), $GA_{19}$ ($0.18{\pm}0.09$), $GA_{20}$ ($0.78{\pm}0.15$), $GA_{24}$ ($0.38{\pm}0.09$), $GA_{34}$ ($0.35{\pm}0.10$), and $GA_{53}$ ($0.17{\pm}0.05$). Plant growth promotion in cucumber, tomato, and young radish plants further evidenced the potential of this strain as a PGP bacterium. The results suggest that GA secretion by L. soli SE134 might prove advantageous for its ameliorative role in crop growth. These findings can be extended for improving the productivity of different crops under diverse environmental conditions.

• Journal of Microbiology and Biotechnology
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• v.16 no.3
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• pp.450-456
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• 2006

Pseudomonas fluorescens MC07 is a growth-promoting rhizobacterium that suppresses mycelial growth in fungi such as Rhizoctonia solani, Pythium ultimum, Fusarium oxysporum, and Phytophthora capsici. To determine the role of the bacterium's antifungal activity in disease suppression, we screened 2,500 colonies generated by Tn5lacZ insertions, and isolated a mutant 157 that had lost antifungal activity. The EcoRI fragment carrying Tn5lacZ was cloned into pBluescript II SK(+) and used as a probe to isolate wild-type clones from a genomic library of the parent strain, MC07. Two overlapping cosmid clones, pEH4 and pEH5, that had hybridized with the mutant clone were isolated. pEH4 conferred antifungal activity to the heterologous host P.fluorescens strain 1855.344, whereas pEH5 did not. Through transposon mutagenesis of pEH4 and complementation analyses, we delineated the 14.7-kb DNA region that is responsible for the biosynthesis of an antifungal compound. DNA sequence analysis of the region identified 11 possible open reading frames (ORF), ORF1 through ORF11. A BLAST search of each putative protein implied that the proteins may be involved in an antifungal activity similar to polyketides.

• Research in Plant Disease
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• v.26 no.1
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• pp.8-18
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• 2020

Red pepper, one of the major economic crops in Korea, is being affected by anthracnose disease caused by Colletotrichum acutatum. To control this disease, an antagonistic bacterial strain, Bacillus subtilis YGB36 identified by 16S rDNA sequencing, physiological and biochemical analyses is used as a biological control agent. In vitro screening revealed that the strain YGB36 possess strong antifungal activity against the pathogen Cylindrocarpon destructans. The strain exhibited cellulase, protease, amylase, siderophore production and phosphate solubility. In vitro conidial germination of C. acutatum was most drastically inhibited by YGB36 cell suspensions (10⁶ cfu/ml) or culture filtrate. Development of anthracnose symptoms was reduced on detached immature green pepper fruits by treatment with cell suspensions, and its control value was recorded as 65.7%. The YGB36 bacterial suspension treatment enhanced the germination rate of red pepper seeds and promoted root development and growth under greenhouse conditions. The in vitro screening of fungicide and insecticide sensitivity test against YGB36 revealed that the bacterial growth was not affected by any of the insecticides, and 11 fungicides out of 21 used. Collectively, our results clearly suggest that the strain YGB36 is considered as one of the potential biocontrol agents against anthracnose disease in red pepper.

• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1001-1010
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• 2005

Bacillus megaterium KL39, an antibiotic-producing plant growth promoting rhizobacterium (PGPR), was selected from soil. The antifungal antibiotic, denoted KL39, was purified from culture filtrate by column chromatography using Dion HP-20, Silica gel, Sephadex LH-20, and prep-HPLC. Thin layer chromatography, employing the solvent system of ethanol:ammonia:water=8:1:1, showed the $R_{f}$. value of 0.32. The antibiotic KL39 showed a negative reaction with ninhydrin solution, positive with iodine vapor, and also positive with Ehrlich reagent. It was soluble in methanol, ethanol, butanol, and acetonitrile, but insoluble in chloroform, toluene, hexane, ethyl ether, or acetone. Its UV spectrum had the maximum absorption at 208 nm. Amino acid composition, FAB-mass, $^{1}H-NMR,\;^{13}C-NMR$, and atomic analyses showed that the antibiotic KL39 (MW=1,071) has a structure very similar to iturin E. The antibiotic KL39 has a broad antifungal spectrum against a variety of plant pathogenic fungi including Rhizoctonia solani, Pyricularia oryzae, Monilinia froeticola, Botrytis cinenea, Altenaria kikuchiana, Fusarium oxysporum, and F. solani. An MIC value of $10\;{\mu}g/ml$ was determined for Phytophthora capsici. Macromolecular incorporation studies with P. capsici using radioactive [$^{3}H-adenine$] as the precursor, indicated that the antibiotic KL39 strongly inhibits the DNA biosynthesis of the fungal cell. Microscopic observation of the antifungal action showed abnormal hyphal swelling of P. capsici. The purified antibiotic KL39 was very effective for the biocontrol of in vivo Phytophthora-blight disease of pepper.

• Microbiology and Biotechnology Letters
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• v.34 no.4
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• pp.311-317
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• 2006

Using PCR amplification, we cloned a cellulase gene (ce/H) from the Bacillus subtilis AH18 which has plant growth-promoting activity and antagonistic ability against pepper blight caused by Phytophthora capsici. The 1.6 kb PCR fragment contained the full sequence of the cellulase gene and the 1,582 bp gene deduced a 508 amino acid sequence. Similarity search in protein database revealed that the cellulase of B. subtilis AH18 was more than 98% homologous in the amino acid sequence to those of several major Bacillus spp. The ce/H was expressed in E. coli under an IPTG inducible lac promoter on the vector, had apparent molecular weight of about 55 kDa upon CMC-SDS-PAGE analysis. Partially purified cellulase had not only cellulolytic activity toward carboxymethyl-cellulose (CMC) but also insoluble cellulose, such as Avicel and filter paper (Whatman No. 1). In addition, the cellulase could degrade a fungal cell wall of Phytophthora capsici. The optimum pH and temperature of the ce/H coded cellulase were determined to be pH 5.0 and $50^{\circ}C$. The enzyme activity was activated by $AgNO_3$ or $CoCl_2$. However its activity was Inhibited by $HgC1_2$. The enzyme activity was activated by hydroxy urea or sodium azide and inhibited by CDTA or EDTA. The results indicate that the cellulase gene, ce/H is an antifungal mechanism of B. subtilis AH18 against phytophthora blight disease in red-pepper.

• Microbiology and Biotechnology Letters
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• v.36 no.4
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• pp.326-335
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• 2008

The siderophore ($siderophore_{AH18}$) of Bacillus subtilis AR18 was determined to be one of catechol type and purified by using Amberlite XAD-2, Sephadex LR-20 chromatography, and reversed-phase RPLC. The $Siderophore_{AH18}$ was identified bacillibactin with its structure by GC-MS, $^1H$-NMR, and $^{13}C$-NMR. $Siderophore_{AH18}$ (bacillibactin) had been confirmed its molecular weight of 883 and chemical structure of $(2,3-dihydroxybenzoate-glycine-threonine)_3$. Purified $siderophore_{AH18}$ showed strong biocontrol ability towards the spore of Phytophthora capsici on PDA and able to effectively suppress (55%) P. capsici causing red-pepper blight in the pot in vivo test.