• Korean Journal of Soil Science and Fertilizer
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• v.43 no.4
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• pp.522-527
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• 2010

Gibberellic acid ($CA_3$) is used in many industries and constitutes the primary gibberellins produced by fungi and bacteria. However, there is no information on $CA_3$ production by Methylobacterium oryzae CBMB20, a novel plant growth promoting bacterium. We investigated the favorable carbon (C) and nitrogen (N) sources and ratios and cultural conditions, such as incubation temperature, pH of the culture medium, and incubation period for the maximum production of $CA_3$ by Methylobacterium oryzae CBMB20. Maximum $CA_3$ production was observed in ammonium mineral salt (AMS) broth supplemented with Na-succinate and $NH_4Cl$ as C and N sources, respectively. The maximum $CA_3$ production was found at the C/N ratio of 5:0.4 g $L^{-1}$. The highest $CA_3$ production was obtained when the bacterial culture was incubated at $30^{\circ}C$ for 96 h at pH 7.

• Korean Journal of Food Science and Technology
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• v.25 no.4
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• pp.366-372
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• 1993

This study mainly designed to high quality of mirin production by using protopast fusion. In order to enhance the acid carboxypeptidase (ACPase) activity by the method of protoplast fusion. In order to enhance the acid carboxypeptidase (ACPase) activity by the method of protopalst fusion, the mutants, Aspergillus oryzae 9-12 and Aspergillus shirosamii IFO 6082-60 were selected by mutation among various mutants. Protoplast of Aspergillus oryzae 9-12 and Aspergillus shirousamii IFO 6082-60 were formed effectively by incubation of the mixtures of chitinase (10mg/ml), cellulase (10mg/ml) and zymolase 20T (5mg/ml). For protopalst fusion, the mixture of two mutant were fused to effective under the optimum conditions by solutions containing 30% PEG 6,000, 0.01M $CaCl_2\;2H_2O$, 0.6M KCl and 0.05M glycine. Fusion frequency was 0.71% and fusant, F-50 appeared ACPase activity of 20,800 unit/g which has 1.5 times higher than that of each mutants.

• Korean Journal Plant Pathology
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• v.3 no.2
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• pp.124-130
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• 1987

The optimum conditions for protoplast formation and regeneration from Pyricularia oryzae Cav. were selected as follows. As a basic solution, 0.02M potassium phosphate buffer solution plus 0.6M KCl adjusted to pH 5.2 with 1N HCl was used. A mixture of enzyme combinations with 20mg Cellulase R-l0/ml, 5mg Macerozyme R-l0/ml and l0mg Driselase/ml used as a lytic enzyme showed better lytie effect than any single enzyme treatment for protoplast formation. Two-day-old mycelia of P. oryzae grown in the mixture of three lytie enzyme solution at $30^{\circ}C$ for 3 hr showed best condition for protoplasts formation. For regeneration from the protoplasts of P. oryzae, potato dextrose agar containing 0.02M potassium phosphate plus 0.6M KCl used as a stabilizer was best for regeneration medium.

• Mycobiology
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• v.47 no.4
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• pp.473-482
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• 2019

Rice blast disease, caused by the ascomycete fungus Magnaporthe oryzae, is one of the most important diseases in rice production. PAS (period circadian protein, aryl hydrocarbon receptor nuclear translocator protein, single-minded protein) domains are known to be involved in signal transduction pathways, but their functional roles have not been well studied in fungi. In this study, targeted gene deletion was carried out to investigate the functional roles of the PAS-containing gene MoPAS1 (MGG_02665) in M. oryzae. The deletion mutant ΔMopas1 exhibited easily wettable mycelia, reduced conidiation, and defects in appressorium formation and disease development compared to the wild type and complemented transformant. Exogenous cAMP restored appressorium formation in ΔMopas1, but the shape of the restored appressorium was irregular, indicating that MoPAS1 is involved in sensing the hydrophobic surface. To examine the expression and localization of MoPAS1 in M. oryzae during appressorium development and plant infection, we constructed a MoPAS1:GFP fusion construct. MoPAS1:GFP was observed in conidia and germ tubes at 0 and 2 h post-infection (hpi) on hydrophobic cover slips. By 8 hpi, most of the GFP signal was observed in the appressoria. During invasive growth in host cells, MoPAS1:GFP was found to be fully expressed in not only the appressoria but also invasive hyphae, suggesting that MoPAS may contribute to disease development in host cells. These results expand our knowledge of the roles of PAS-containing regulatory genes in the plant-pathogenic fungus M. oryzae.

• The Plant Pathology Journal
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• v.32 no.1
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• pp.8-15
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• 2016

To identify a novel biopesticide controlling rice blast disease caused by Magnaporthe oryzae, 700 plant extracts were evaluated for their inhibitory effects on mycelial growth of M. oryzae. The L. foenum-graecum Herba extract showed the lowest inhibition concentration ($IC_{50}$) of $39.28{\mu}g/ml$, which is lower than the $IC_{50}$ of blasticidin S ($63.06{\mu}g/ml$), a conventional fungicide for rice blast disease. When treatments were combined, the $IC_{50}$ of blasticidin S was dramatically reduced to $10.67{\mu}g/ml$. Since ABC transporter genes are involved in fungicide resistance of many organisms, we performed RT-PCR to investigate the transcriptional changes of 40 ABC transporter family genes of M. oryzae treated with the plant extract, blasticidin S, and tetrandrine, a recognized ABC transporter inhibitor. Four ABC transporter genes were prominently activated by blasticidin S treatment, but were suppressed by combinational treatment of blasticidin S with the plant extract, or with tetrandrine that didn't show cellular toxicity by itself in this study. Mycelial death was detected via confocal microscopy at 24 h after plant extract treatment. Finally, subsequent rice field study revealed that the plant extract had high control efficacy of 63.3% and should be considered a biopesticide for rice blast disease. These results showed that extract of L. foenum graecum Herba suppresses M. oryzae ABC transporter genes inducing mycelial death and therefore may be a potent novel biopesticide.

• The Plant Pathology Journal
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• v.30 no.3
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• pp.304-309
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• 2014

The rpf genes and $colS_{XOO1207}/colR_{XOO1208}$ were known to require for virulence of Xanthomonas oryzae pv. oryzae (Xoo). In Xoo KACC10331 genome, two more colS/colR genes, $colS_{XOO3534}$ (raxH)/$colR_{XOO3535}$ (raxR) and $colS_{XOO3762}/colR_{XOO3763}$ were annotated. The $colS_{XOO3534}/colR_{XOO3535}$ were known to control AvrXa21 activity and functions of $colS_{XOO3762}/colR_{XOO3763}$ were unknown in Xoo. To characterize the relationship between rpf and colS/colR genes, expression of colS/colR genes in Rpf mutants of Xoo were analyzed with quantitative reverse transcription PCR (qRT-PCR). Expressions of all three colS/colR genes increased in the rpfF mutant in which DSF synthesis is defective. Expression of $colS_{XOO1207}/col-R_{XOO1208}$, $colS_{XOO3534}/colR_{XOO3535}$ and $colS_{XOO3762}/colR_{XOO3763}$ increased 2, 2-7, 3-13 folds respectively. Expression of $colS_{XOO3534}$ and $colS_{XOO3762}$ also increased 2-4 folds in the rpfG mutant in which the signal from DSF is no longer transferred to down-stream. Expression of the other colS/colR genes was not significantly changed in the rpfG mutant compared to the wild type. Since RpfF and RpfG are responsible for DSF synthesis and signal transfer from DSF to down-stream to regulate virulence gene expression, these results suggest that the DSF and DSF-mediated signal regulate negatively three colS/colR genes in Xoo.

• Journal of Applied Biological Chemistry
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• v.58 no.3
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• pp.253-258
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• 2015

Xanthomonas oryzae pv. oryzae (Xoo), X. campestris pv. vesicatoria (Xcv), and Pectobacterium carotovorum pv. carotovorum (Pcc) are the causative agents of bacterial blight in rice, bacterial spot in pepper, and bacterial soft rot in carrot and cabbage, respectively. To isolate novel microbial extracts with antimicrobial activities against these bacteria, approximately 5,300 different Streptomyces extracts were prepared and tested. Microbial cultures from various Streptomyces strains isolated from the Jeju Island, Baekam, Mankyoung river, Jiri mountain etc. in Korea were extracted into three different factions -secreted hydrophobic, secreted hydrophilic, and mycelia- using ethyl acetate, water, and methanol. Initially, 34, 29, and 10 extracts were selected as having antibacterial activities against Xoo, Xcv, and Pcc, respectively. Extracts 1169G4, 1172E9, and 1172E10 had the highest growth inhibition activities against both Xoo and Xcv, and extracts 1151H7 and 1152H7 showed the highest growth inhibition activities against Pcc.

• Korean Journal of Food Science and Technology
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• v.24 no.5
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• pp.477-481
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• 1992

As a series of fundamental research projects to produce doenjang (Korean fermented soy paste) of better quality, two kinds of doenjang were manufactured from a traditional meju (Korean soy bean koji) and the mixed with Aspergillus oryzae and Bacillus natto, and histological changes in the cell structures of soy bean of the two were reported doenjang samples were observed and compared during the entire period of fermentation processes. Cell walls of the soy bean were ruptured by pressure and heat during the pressure cooking process and some of them were observed to have the ghost-like shapes. Remarkable differences in the plasmolysis of the cytoplasms were observed between the seed coat and the inner part of soy bean. Small vacuoles resulting from the fusion of the glycoprotein globules by protease and from the hydrolysis of the starch granules by amylase were also observed. Penetration of microorganisms was transferred from the seed coat to the inside of soy bean as the fermentation proceeded. Slimy substances were observed on the seed coat and the parenchyma cells of soy bean fermented with the mixed with Aspergillus oryzae and Bacillus natto. Cell walls of soy bean became difficult to stain and they showed unusual, polygonal shapes as the fermentation proceeded. Samples fermented with the mixed with Aspergillus oryzae and Bacillus natto showed more remarkable tendencies than traditional meju.

• Journal of Microbiology
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• v.43 no.6
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• pp.475-486
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• 2005

Fungal secondary metabolites constitute a wide variety of compounds which either playa vital role in agricultural, pharmaceutical and industrial contexts, or have devastating effects on agriculture, animal and human affairs by virtue of their toxigenicity. Owing to their beneficial and deleterious characteristics, these complex compounds and the genes responsible for their synthesis have been the subjects of extensive investigation by microbiologists and pharmacologists. A majority of the fungal secondary metabolic genes are classified as type I polyketide synthases (PKS) which are often clustered with other secondary metabolism related genes. In this review we discuss on the significance of our recent discovery of chalcone synthase (CHS) genes belonging to the type III PKS superfamily in an industrially important fungus, Aspergillus oryzae. CHS genes are known to playa vital role in the biosynthesis of flavonoids in plants. A comparative genome analyses revealed the unique character of A. oryzae with four CHS-like genes (csyA, csyB, csyC and csyD) amongst other Aspergilli (Aspergillus nidulans and Aspergillus fumigatus) which contained none of the CHS-like genes. Some other fungi such as Neurospora crassa, Fusarium graminearum, Magnaporthe grisea, Podospora anserina and Phanerochaete chrysosporium also contained putative type III PKSs, with a phylogenic distinction from bacteria and plants. The enzymatically active nature of these newly discovered homologues is expected owing to the conservation in the catalytic residues across the different species of plants and fungi, and also by the fact that a majority of these genes (csyA, csyB and csyD) were expressed in A. oryzae. While this finding brings filamentous fungi closer to plants and bacteria which until recently were the only ones considered to possess the type III PKSs, the presence of putative genes encoding other principal enzymes involved in the phenylpropanoid and flavonoid biosynthesis (viz., phenylalanine ammonia-lyase, cinnamic acid hydroxylase and p-coumarate CoA ligase) in the A. oryzae genome undoubtedly prove the extent of its metabolic diversity. Since many of these genes have not been identified earlier, knowledge on their corresponding products or activities remain undeciphered. In future, it is anticipated that these enzymes may be reasonable targets for metabolic engineering in fungi to produce agriculturally and nutritionally important metabolites.

• Journal of Microbiology and Biotechnology
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• v.27 no.2
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• pp.277-288
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• 2017

Rhizomucor miehei NRRL 5282 and Rhizopus oryzae NRRL 1526 can produce lipases with high synthetic activities in wheat bran-based solid-state culture. In this study, the purification and biochemical characterization of the lipolytic activities of these lipases are presented. SDS-PAGE indicated a molecular mass of about 55 and 35 kDa for the purified R. miehei and Rh. oryzae enzymes, respectively. p-Nitrophenyl palmitate (pNPP) hydrolysis was maximal at $40^{\circ}C$ and pH 7.0 for the R. miehei lipase, and at $30^{\circ}C$ and pH 5.2 for the Rh. oryzae enzyme. The enzymes showed almost equal affinity to pNPP, but the $V_{max}$ of the Rh. oryzae lipase was about 1.13 times higher than that determined for R. miehei using the same substrate. For both enzymes, a dramatic loss of activity was observed in the presence of 5 mM $Hg^{2+}$, $Zn^{2+}$, or $Mn^{2+}$, 10 mM N-bromosuccinimide or sodium dodecyl sulfate, and 5-10% (v/v) of hexanol or butanol. At the same time, they proved to be extraordinarily stable in the presence of n-hexane, cyclohexane, n-heptane, and isooctane. Moreover, isopentanol up to 10% (v/v) and propionic acid in 1 mM concentrations increased the pNPP hydrolyzing activity of R. miehei lipase. Both enzymes had 1,3-regioselectivity, and efficiently hydrolyzed p-nitrophenyl (pNP) esters with C8-C16 acids, exhibiting maximum activity towards pNP-caprylate (R. miehei) and pNP-dodecanoate (Rh. oryzae). The purified lipases are promising candidates for various biotechnological applications.