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Usability of DNA Sequence Data: from Taxonomy over Barcoding to Field Detection. A Case Study of Oomycete Pathogens

  • Choi, Young-Joon;Thines, Marco
    • 한국균학회소식:학술대회논문집
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    • 2015.11a
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    • pp.41-41
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    • 2015
  • Oomycetes belong to the kingdom Straminipila, a remarkably diverse group which includes brown algae and planktonic diatoms, although they have previously been classified under the kingdom Fungi. These organisms have evolved both saprophytic and pathogenic lifestyles, and more than 60% of the known species are pathogens on plants, the majority of which are classified into the order Peronosporales (includes downy mildews, Phytophthora, and Pythium). Recent phylogenetic investigations based on DNA sequences have revealed that the diversity of oomycetes has been largely underestimated. Although morphology is the most valuable criterion for their identification and diversity, morphological species identification is time-consuming and in some groups very difficult, especially for non-taxonomists. DNA barcoding is a fast and reliable tool for identification of species, enabling us to unravel the diversity and distribution of oomycetes. Accurate species determination of plant pathogens is a prerequisite for their control and quarantine, and further for assessing their potential threat to crops. The mitochondrial cox2 gene has been widely used for identification, taxonomy and phylogeny of various oomycete groups. However, recently the cox1 gene was proposed as a DNA barcode marker instead, together with ITS rDNA. To determine which out of cox1 or cox2 is best suited as universal oomycete barcode, we compared these two genes in terms of (1) PCR efficiency for 31 representative genera, as well as for historic herbarium specimens, and (2) in terms of sequence polymorphism, intra- and interspecific divergence. The primer sets for cox2 successfully amplified all oomycete genera tested, while cox1 failed to amplify three genera. In addition, cox2 exhibited higher PCR efficiency for historic herbarium specimens, providing easier access to barcoding type material. In addition, cox2 yielded higher species identification success, with higher interspecific and lower intraspecific divergences than cox1. Therefore, cox2 is suggested as a partner DNA barcode along with ITS rDNA instead of cox1. Including the two barcoding markers, ITS rDNA and cox2 mtDNA, the multi-locus phylogenetic analyses were performed to resolve two complex clades, Bremia lactucae (lettuce downy mildew) and Peronospora effuse (spinach downy mildew) at the species level and to infer evolutionary relationships within them. The approaches discriminated all currently accepted species and revealed several previously unrecognized lineages, which are specific to a host genus or species. The sequence polymorphisms were useful to develop a real-time quantitative PCR (qPCR) assay for detection of airborne inoculum of B. lactucae and P. effusa. Specificity tests revealed that the qPCR assay is specific for detection of each species. This assay is sensitive, enabling detection of very low levels of inoculum that may be present in the field. Early detection of the pathogen, coupled with knowledge of other factors that favor downy mildew outbreaks, may enable disease forecasting for judicious timing of fungicide applications.

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The Kinetics of Non-Enzymatic Browning Reaction in Green Tea During Storage at Different Water Activities and Temperatures (온도와 수분활성을 달리한 녹차 저장중의 비효소적 갈변)

  • Kim, Young-Suk;Jung, Yeon-Hwa;Chun, Soon-Sil;Kim, Mu-Nam
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.17 no.3
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    • pp.226-232
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    • 1988
  • Non-enzymatic browning is a carbohydrate dehydration reaction, accelerated thorough the interaction of amino compounds. Reaction depends on several factors including temperature, reactant concentration, pH, water activity and specific ion concentrations, and result in progressive development of brown pigments in the affected food systems. The present study was designed to utilize a kinetic approach to analyze the effect of temperature and water activity on the browning development in green ten. The green tea was controlled at aw of 0.33, 0.44, 0.52 and 0.65 using saturated salt solutions and then stored at 35,45 and $55^{\circ}C$. Author portion of the sample of which the water activities were controlled in the same manner was stored at 35 and $55^{\circ}C$ alternately with 7 days interval. Simplified kinetic models were used to obtain the various kinetic parameters for browning development in green tea subjected to accelerated shelf-life tests(ASLT). The reaction of browning development was zero order. The activation energies calculated from Arrhenius plot ranged $1.5{\sim}2.4kcal/mole$ and $Q_{10}$ values were between 1.07 and 1.12. These kinetic parameters were then used to predict browning development under the nonsteady storage. Assessed from the parameters the shelf-lives at $25^{\circ}C$, the time to reach 1.02 O.D./g solid at which severe brown color change could be detectable, ranged 57 to 113 days and showed decrease with increase in aw. The predicted shelf-lives at different water activities were a little higher than actual values.

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Functional Analysis of Bacillus subtilis Isolates and Biological Control of Red Pepper Powdery Mildew Using Bacillus subtilis R2-1 (Bacillus subtilis 균주의 기능성 분석 및 이를 활용한 고추 흰가루병 방제)

  • Kim, Yong-Ki;Hong, Sung-Jun;Shim, Chang-Ki;Kim, Min-Jeong;Choi, Eun-Jung;Lee, Min-Ho;Park, Jong-Ho;Han, Eun-Jung;An, Nan-Hee;Jee, Hyeong-Jin
    • Research in Plant Disease
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    • v.18 no.3
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    • pp.201-209
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    • 2012
  • The multi-function of 18 Bacillus subtilis isolates collected from agricultural extension centers of local government and National Academy of Agricultural Science was investigated by measuring their antifungal activities against five plant pathogens, such as Rhizoctonia solani, Colletotrichum acutatum, Fusarium oxysporum, Magnaporthe oryzae and Phytophthora capsici, phosphorus solubilization ability, production of indole acetic acid (IAA) and siderophore, and nitrogen fixation. The B. subtilis isolates showed antifungal activity against several plant pathogens and nitrogen fixation activity, and produced siderophore and IAA. They could control pepper powdery mildew (Leveillula taurica), but there was no difference in control efficacy among the B. subtilis isolates. In fields, the control efficacy of B. subtilis R2-1 ($10^8$ cells/ml) was compared with two microbial fungicides, Q-pect and Topsid. In 2009, the control efficacy of B. subtilis R2-1 (37.7%) was lower than that of Topsid (47.6%), but higher than that of Q-pect (25.7%). In 2010, the control efficacy of B. subtilis R2-1 (83.3%) was higher than that of Topsid (67.9%). In order to elucidate mode of action of B. subtilis R2-1 for controlling pepper powdery mildew, spore germination rates of pepper powdery mildew pathogen collected on treated leaves was investigated when suspensions of B. subtilis R2-1 and two microbial fungicides (Q-pect and Topsid) were foliar-sprayed. They highly suppressed spore germination of the pathogen with inhibition values of 84.2% for B. subtilis R2-1, 97.9% for Q-pect and 94.7% for Topsid. Further study on the mass-culturing method and formulation is needed for development of a microbial fungicide.

Studies on Fine Spirits Aging [Part I]-On the Aptitude of the Korean Oak Varieties as Barrels for Aging Apple Fine Spirits- (증류주(蒸溜酒) 숙성(熟成)에 관(關)한 연구(硏究) 제1보[第一報]-사과 증류주(蒸溜酒) 숙성(熟成)에 있어서 숙성통재(熟成桶材)로서 한국산(韓國産) 참나무 품종별(品種別) 이용적성(利用適性)에 관(關)하여-)

  • Lee, Ke-Ho
    • Applied Biological Chemistry
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    • v.20 no.1
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    • pp.66-80
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    • 1977
  • This research was carried as a part of the basic study, in which the aptitude of theKorean oak varieties as barrels for aging apple fine spirits was investigated, and thefollowing results were obtained. 1. Following was the result of the chemical analysis of the fruits which are now mass-produced and can be used as a substitute for raw materials for wine production. Apple (Malus pumila Miller var. domestica Schneider) : Total sugar. total acid, volatile acid and pectin of Jonathan (Hong-og) were 13.95%, 0.46%, 0.012%, 0.20% respectively. Total sugar, total acid, volatile acid and pectin of Ralls (Koog-kwang) were 13.35%, 0.43%, 0.011%, 0.45% respectively. 2. Because of low yield of apple juice due to cellulose, pectin, hemicellulose which are present besides sugars, acids in apples, the apple juice were treated with xylanase of Aspergillus niger SUAFM-430, cellulase and pectinase of Aspergillus niger SUAFM-6. This treatment increased the yield of apple juice. And the apple juice was sterilized by adding potassium metabisulfite $(K_2S_20_5)$ and Saccharomyces cerevisae var. ellipsoideus Rasse Johannisberg II (SUAFM-1018) as a cultivation yeast, which has a strong fermentation power was used to ferment. The yield of apple wine based on raw material was 86-87%. The amount of ethanol, extract and methanol obtained from Jonathan and Ralls were 13.5%, 5.4%, 0.04-0.05% respectively. 3. Wines were distilled for two times by the pot still method to make fine spirits. The yield of fine spirits from apple wine mash was 86.6%, and the pH of fine spirits from Jonathan and Ralls were 4.1, 4.2 respectively. 4. The oak chips made of inner part or outer part of 24 Korean oak varieties were used to select the barrel for aging fine spirits. Two oak chips (one oak chip: $1{\times}1{\times}5cm$) of the inner part or of the outer part of each oak variety were dipped into 300 ml of fine spirits, which was bottled in 640ml beer bottle, and followed aging. The colors, flavors and tastes of the fine spirits were checked during 6 months. A. As a criterion for the first screening of oak barrels for aging fine spirits, the rate five of color extraction was determined. The oak chips showed good results in their order as follows and the best 5 varieties were selected. Gal-cham: Quercus aliena Blume (Inner part), Gul-cham: Quercus variabilis Blume (Outer part), Gal-chain: Quercus aliena Blume (Outer part), Jol-cham: Quercus serrata Thumb (Inner and Outer part). Sin-gal-cham: Quercus mongolica Fisher (Outer and Inner part) Sang-su-ri: Quercus acutissima Carruthers (Outer and Inner part) B. To find out the influence of aging temperature on aging, apple fine spirits were aged by dipping each oak chip at room temperature $(24-25^{\circ}C)$) and $45^{\circ}C$. Aging at $45^{\circ}C$ gave the best result followed aging at $30^{\circ}C$ and then at room temperature. C. Apple fine spirits was aged for six months by dipping oak chips in Erlenmeyer flasks and was irradiated with U.V light. The U.V irradiation enhanced the aging effect by nearly two times, compared with the aging without U.V irradiation. D. In aging apple fine spirits by dipping two oak chips, it was observed that the extent of the extraction of most components of oak chips were strongly dependent upon the pH of fine spirits. E. Oak chips of five selected oak varieties and a Limousin white oak from France as a control were used. Each apple fine spitits was dipped by two oak chips, and was aged at room temperature $(24-25^{\circ}C)$, $30^{\circ}C$, $45^{\circ}C$, and with the U.V irradiation at room temperature shaking every week. After six months of aging, the panel test of these aged fine spirits (Young Brandy) showed the following result. Young brandy of apples aged at $45^{\circ}C$ by dipping oak chips of Gal-chain was almost as the fine spirits which were aged at room temperature by dipping Limousin white oak chips from France. Young brandy of with U.V. irradiation at room temperature which were aged by dipping oak chips of Gal-chain was a little worse than that from the fine spirits aged at room temperature by dipping Limousin white oak chips from France. And so, Korean oak varieties are thought to be able to be used for aging every apple fine spirit which was here investigated.

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Assessment of Heavy Metal Effects on the Freshwater Microalga, Chlorella vulgaris, by Chlorophyll Fluorescence Analysis (엽록소형광분석을 이용한 담수산 클로렐라(Chlorella vulgaris)에 미치는 중금속의 영향 평가)

  • Oh, Soon-Ja;Koh, Seok-Chan
    • Journal of Environmental Science International
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    • v.24 no.12
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    • pp.1591-1600
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    • 2015
  • The response of the freshwater microalga, Chlorella vulgaris, to heavy metal stress was examined based on chlorophyll fluorescence analysis to assess the toxic effects of heavy metals in freshwater ecosystems. When toxic effects were analyzed using regular chlorophyll fluorescence analysis, photosystem II activity($F_v/F_m$) decreased significantly when exposed to $Cu^{2+}$ and $Hg^{2+}$ for 12 h, and decreased in the order of $Hg^{2+}>Cu^{2+}>Cd^{2+}>Ni^{2+}$ when exposed for 24h. The effective photochemical quantum yield(${\phi}{\prime}_{PSII}$), chlorophyll fluorescence decrease ratio($R_{Fd}$), minimal fluorescence yield($F_o$), and non-photochemical quenching(NPQ), but not photochemical quenching(qP), responded sensitively to $Hg^{2+}$, $Cu^{2+}$, and $Cd^{2+}$. These results suggest that $F_v/F_m$, as well as ${\phi}{\prime}_{PSII}$, $R_{Fd}$, $F_o$, and NPQ could be used to assess the effects of heavy metal ions in freshwater ecosystems. However, because many types of heavy metal ions and toxic compounds co-occur under natural conditions, it is difficult to assess heavy metal toxicity in freshwater ecosystems. When Chlorella was exposed to heavy metal ions for 12 or 24h, $F_v/F_m$ and maximal fluorescence yield($F_m$) changed in response to $Hg^{2+}$ and $Cu^{2+}$ based on image analysis. However, assessing quantitatively the toxic effects of several heavy metal ions is challenging.

17Beta-Estradiol Regulates NUCB2/Nesfatin-1 Expression in Mouse Oviduct

  • Sun, Sojung;Shin, Jungwoo;Jang, Jiwon;Hwang, Seungyeon;Kim, Jeongwoo;Kong, Jinseong;Yang, Hyunwon
    • Development and Reproduction
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    • v.24 no.1
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    • pp.43-52
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    • 2020
  • NUCB2/nesfatin-1 known to regulate appetite and energy homeostasis is expressed not only in the hypothalamus, but also in various organs and tissues. Our previous reports also demonstrated that NUCB2/nesfatin-1 was expressed in the reproductive organs, including the ovaries, uterus, and testes of mice. However, it is yet known whether NUCB2/nesfatin-1 is expressed in the oviduct and how its expression is regulated. Therefore, we investigated the expression of NUCB2/nesfatin-1 in the oviduct and its expression is regulated by gonadotropin. Immunohistochemical staining results showed that nesfatin-1 protein was localized in epithelial cells of the oviduct. As a result of quantitative real-time PCR (qRT-PCR) and Western blot, NUCB2/nesfatin-1 was detected strongly in the oviducts. During the estrus cycle, NUCB2/nesfatin-1 expression in the oviducts was markedly higher in the proestrus stage than in other estrus stages. In order to elucidate whether the expression of NUCB2 mRNA is controlled by the gonadotropins, we injected PMSG and hCG and measured NUCB2 mRNA level in the oviduct after injection. Its level was increased in the oviduct after PMSG injection, but no significant change after hCG injection. In addition, NUCB2 mRNA levels were markedly reduced after ovariectomy, while recovered after 17β-estradiol (E2) injection, but not by progesterone (P4). This study demonstrated that NUCB2/nesfatin-1 is highly expressed in the oviduct of mouse and its expression is regulated by E2 secreted by the ovaries. These results suggest that NUCB2/nesfatin-1 expressed by the oviduct may affect the function of the oviduct regulated by the ovaries.

Expression, Purification, and Characterization of C-Terminal Amidated Glucagon in Streptomyces lividans

  • Qi, Xiaoqiang;Jiang, Rong;Yao, Cheng;Zhang, Ren;Li, Yuan
    • Journal of Microbiology and Biotechnology
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    • v.18 no.6
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    • pp.1076-1080
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    • 2008
  • Glucagon, a peptide hormone produced by alpha-cells of Langerhans islets, is a physiological antagonist of insulin and stimulator of its secretion. In order to improve its bioactivity, we modified its structure at the C-terminus by amidation catalyzed by a recombinant amidase in bacterial cells. The human gene coding for glucagon-gly was PCR amplified using three overlapping primers and cloned together with a rat ${\alpha}$-amidase gene in plasmid pMGA. Both genes were expressed under control of the strong constitutive promoter of aph and secretion signal melC1 in Streptomyces lividans. With Phenyl-Sepharose 6 FF, Q-Sepharose FF, SP-Sepharose FF chromatographies and HPLC, the peptide was purified to about 93.4% purity. The molecular mass of the peptide is 3.494 kDa as analyzed by MALDI TOF, which agrees with the theoretical mass value of the C-terminal amidated glucagon. The N-terminal sequence of the peptide was also determined, confirming its identity with human glucagon at the N-terminal part. ELISA showed that the purified peptide amide is bioactive in reacting with glucagon antibodies.

Low Temperature Sintering and Piezoelectric Properties of PCW-PMN-PZT Ceramics with the Variation of Sintering Aids (소결조제 변화에 따른 PCW-PMN-PZT세라믹스의 저온소결 및 압전특성)

  • Chung, Kwang-Hyun;Lee, Duck-Chool;Yoo, Ju-Hyun
    • Journal of the Korean Institute of Electrical and Electronic Material Engineers
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    • v.17 no.12
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    • pp.1320-1325
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    • 2004
  • In this study, in order to develop the low temperature sintering ceramics for multilayer piezoelectric transformer, PCW-PMN-PZT ceramics added with Li$_2$CO$_3$, Bi$_2$O$_3$ and CuO as sintering aids were manufactured, and their microstructural, dielectric and piezoelectric properties were investigated. When the only CuO was added, specimens could not be sintered below 98$0^{\circ}C$. However, when Li$_2$CO$_3$ and Bi$_2$O$_3$ were added, specimens could be sintered below 98$0^{\circ}C$. Li$_2$CO$_3$ and Bi$_2$O$_3$ addition were proved to lower sintering temperature of piezoelectric ceramics due to the effect of Li$_2$O-Bi$_2$O$_3$ liquid phase. Li$_2$CO$_3$ and Bi$_2$O$_3$ added specimens showed higher piezoelectric properties than those of the only CuO added specimens. At 0.2 wt% Li$_2$CO$_3$ and 0.3 wt% Bi$_2$O$_3$ added specimen sintered at 92$0^{\circ}C$, the dielectric constant of 1457, electromechanical coupling factor of 0.56 and mechanical quality factor of 1000 were shown, respectively. These values are suitable for multilayer piezoelectric transformer application.

Altered Proteome of Extracellular Vesicles Derived from Bladder Cancer Patients Urine

  • Lee, Jingyun;McKinney, Kimberly Q.;Pavlopoulos, Antonis J.;Niu, Meng;Kang, Jung Won;Oh, Jae Won;Kim, Kwang Pyo;Hwang, Sunil
    • Molecules and Cells
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    • v.41 no.3
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    • pp.179-187
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    • 2018
  • Proteomic analysis of extracellular vesicles (EVs) from biological fluid is a powerful approach to discover potential biomarkers for human diseases including cancers, as EV secreted to biological fluids are originated from the affected tissue. In order to investigate significant molecules related to the pathogenesis of bladder cancer, EVs were isolated from patient urine which was analyzed by mass spectrometry based proteomics. Comparison of the EV proteome to the whole urine proteome demonstrated an increased number of protein identification in EV. Comparative MS analyses of urinary EV from control subjects and bladder cancer patients identified a total of 1,222 proteins. Statistical analyses provided 56 proteins significantly increased in bladder cancer urine, including proteins for which expression levels varied by cancer stage (P-value < 0.05). While urine represents a valuable, non-invasive specimen for biomarker discovery in urologic cancers, there is a high degree of intra- and inter-individual variability in urine samples. The enrichment of urinary EV demonstrated its capability and applicability of providing a focused identification of biologically relevant proteins in urological diseases.

Manure Based Duckweed Production in Shallow Sink : Effect of Genera on Biomass and Nutrient Yield of Duckweed under the Same Nutritional and Management Conditions

  • Chowdhury, S.A.;Sultana, N.;Huque, K.S.;Huque, Q.M.E.
    • Asian-Australasian Journal of Animal Sciences
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    • v.13 no.5
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    • pp.686-693
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    • 2000
  • Biomass yield, nutritive value and nutrient utilization efficiency of different genera of duckweed (DW, Lemnaceae) under the same nutritional and management conditions were studied. Three genera of DW namely: Lemna perpusilla, Spirodela polyrhiza and Woljfia arrhiza, were cultivated in 18 plastic sinks each of $59{\times}54{\times}35cm^3$ size. Each sink contained approximately 80 L of water and was charged once with 6.648 kg of anaerobically fermented cow dung effluent. The seed rate for Spirodela, Lemna and Wolffia were 600, 400 and $600g/m^2$ respectively. Duckweed were harvested at 48 hours intervals. Media total N concentration for Lemna and Wolffia gradually increased with time. Growth of duckweed was measured by subtracting the inoculum from the total biomass production. Growth of Spirodela ceased within 4 days but Lemna and Wolffia continue to grow up to 34 days. Bio-mass yield was significantly (p<0.05) higher in Wolffia (906 kg/ha/d) than Lemna (631 kg/ha/d). Dry matter (DM) yield was very similar in Lemna (14.80 kg/ha/d) and Wolffia (14.57 kg/ha/d). The N content was non-significant higher in Lemna (5.45%) than Wolffia (5.00%) and Spirodela (4.6%). The crude protein (CP, $N{\times}6.25$) yield was non-significantly higher in Lemna (4.83 kg/ha/d) than Wolffia (4.32 kg/ha/d). The acid detergent fibre (ADF) content was the highest in Wolffia (28.59%), followed by Spirodela (19.47%) and Lemna (12.39%). Utilization efficiency of CP was 273 and 314% respectively for Wolffia and Lemna. However, similar efficiency values for Spirodela was only 1.5%. Considering the bio-mass yield, nutritive value and nutrient utilization efficiency, production performance of DW were in the order of Lemna>Wolffia>Spirodela under the present experimental conditions.