Laixin Luo

Determination of tobramycin in soil by HPLC with ultrasonic-assisted extraction and solid-phase extraction

Pharmaceuticals residues in the environment have become a growing scientific interest worldwide. In the light of the possible harmful effects of tobramycin, a rapid and sensitive analytical method for determination of tobramycin in soil was developed. The extraction and purification methods, derivatization conditions, and chromatographic conditions in the determination of tobramycin in soil have been fully investigated. Extraction was carried out by a combination of vortex mixer and ultrasonic oscillation using acetone/water as the extraction agent. The extract was concentrated to 1 mL and passed through the C(18) SPE cartridge rinsed with water (3 mL), methanol (3 mL). The derivatization procedure was followed by the reaction of tobramycin with 4-Chloro-3,5-dinitrobenzotrifluoride at 60°C for 10 min in pH 9.0 H(3)BO(3)-Na(2)B(4)O(7) medium. The labeled tobramycin was determined by high performance liquid chromatography at 245 nm. Separation was accomplished within 15 min in gradient elution mode with trifluoroacetic acid in mobile phase as ion-pair reagent. The correlation coefficient for the method was 0.9999 in concentrations ranging from 0.10 to 100.0 μg/g. The limit of detection was 0.02 μg/g for tobramycin in soil at a signal-to-noise ratio of 3. The calculated recoveries of the proposed method were from 78.0 to 91.0% and RSDs were 3.38-9.79% in the application to the quantitative determination of tobramycin in all types of soil. The method will help to establish adequate monitoring of tobramycin residue in soil and make the contribution to environmental behavior evaluation.

Biofumigation on Post-Harvest Diseases of Fruits Using a New Volatile-Producing Fungus of Ceratocystis fimbriata

A variety of volatile organic compounds (VOCs) produced by Ceratocystis fimbriata have strong bioactivity against a wide range of fungi, bacteria and oomycetes. Mycelial growth, conidial production, and spore germination of fungi and oomycetes were significantly inhibited after exposure to cultures of C. fimbriata, and colony formation of bacteria was also inhibited. Two post-harvest diseases, peach brown rot caused by Monilinia fructicola and citrus green mold caused by Penicillium digitatum, were controlled during a 4-day storage by enclosing wound-inoculated fruits with 10 standard diameter Petri plate cultures of C. fimbriata in a 15 L box. The fruits were freshly inoculated at onset of storage and the cultures of C. fimbriata were 6 days old. Percentage of control was 92 and 97%, respectively. After exposure to C. fimbriata VOCs, severely misshapen hyphae and conidia of these two post-harvest pathogens were observed by scanning electron microscopy, and their pathogenicity was lost or greatly reduced.

Development and validation of a HPLC method for determination of levonorgestrel and quinestrol in rat plasma

Levonorgestrel and quinestrol, commonly known as EP-1, has long been used in the control of wild rodents. Up to the present time, however, no method for simultaneous quantification of levonorgestrel and quinestrol in rat plasma has been reported. In the present study, a sensitive reverse-phase high-performance liquid chromatography with ultraviolet detection (RP-HPLC-UV) method for quantification of levonorgestrel and quinestrol in rat plasma has been developed. It uses a Kromasil ODS C(18) column and acetonitrile-0.1% formic acid (85 : 15, v/v) mobile phase at ambient temperature. The plasma sample was prepared by hexane-isoamyl alcohol extraction (90 : 10, v/v). The flow rate and detection wavelength were 1.0 mL/min and 230 nm. The correlation coefficients were greater than 0.9995 within 0.08-50 microg/mL for levonorgestrel and 0.12-50 microg/mL for quinestrol, and the limits of detection were 0.02 and 0.05 microg/mL for levonorgestrel and quinestrol, respectively. Average recovery ranged from 92.5 to 96.3% and inter-day RSDs were less than 7.56%. This method can be applied to the further pharmacokinetic study of levonorgestrel and quinestrol in rat plasma.

Identifying optimal reference genes for the normalization of microRNA expression in cucumber under viral stress

Cucumber green mottle mosaic virus (CGMMV) is an economically important pathogen and causes significant reduction of both yield and quality of cucumber (Cucumis sativus). Currently, there were no satisfied strategies for controlling the disease. A better understanding of microRNA (miRNA) expression related to the regulation of plant-virus interactions and virus resistance would be of great assistance when developing control strategies for CGMMV. However, accurate expression analysis is highly dependent on robust and reliable reference gene used as an internal control for normalization of miRNA expression. Most commonly used reference genes involved in CGMMV-infected cucumber are not universally expressed depending on tissue types and stages of plant development. It is therefore crucial to identify suitable reference genes in investigating the role of miRNA expression. In this study, seven reference genes, including Actin, Tubulin, EF-1α, 18S rRNA, Ubiquitin, GAPDH and Cyclophilin, were evaluated for the most accurate results in analyses using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gene expression was assayed on cucumber leaves, stems and roots that were collected at different days post inoculation with CGMMV. The expression data were analyzed using algorithms including delta-Ct, geNorm, NormFinder, and BestKeeper as well as the comparative tool RefFinder. The reference genes were subsequently validated using miR159. The results showed that EF-1α and GAPDH were the most reliable reference genes for normalizing miRNA expression in leaf, root and stem samples, while Ubiquitin and EF-1α were the most suitable combination overall.

Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR

Clavibacter michiganensis subsp. michiganensis (Cmm) is a seed-borne pathogen that causes bacterial canker disease of tomato. Cmm is typically detected in tomato seeds using quantitative real-time polymerase chain reaction (qPCR) combined with culture-based isolation. The viable but nonculturable (VBNC) state of Cmm may result in the underestimation or false negative detection of the pathogen. In the present study, propidium monoazide (PMA) and its improved structure PMAxx were used to pretreat Cmm prior to DNA extraction, followed by qPCR. Both PMA and PMAxx could bind to the chromosomal DNA of dead bacterial cells and therefore block DNA amplification by PCR. This effect, however, does not occur in living bacterial cells, as the chemicals cannot penetrate through the undamaged cell membrane. Both viable and dead Cmm cells were treated with PMA and PMAxx at various concentrations. With this treatment, the range of the cell population was determined for effective detection. PMAxx showed a better discrimination effect than PMA on the viable and dead cells of Cmm and was therefore used throughout the present study. VBNC cells of Cmm (108 CFU mL-1) was induced by 50 μM copper sulfate, which was detected at different sampling times up to a month by using both PMAxx-qPCR and flow cytometry assays. The optimal PMAxx concentration was 20 μM for detecting membrane-intact Cmm cells. High specificity and sensitivity were obtained at Cmm concentrations ranging from 103 to 107 CFU mL-1. The accurate and robust results of PMAxx-qPCR were confirmed by flow cytometry method to detect viable Cmm cells. Furthermore, the PMAxx-qPCR assay was successfully used in detecting VBNC Cmm cells in tomato seeds with as few as 10 seeds per set.

Roles of Genotype-Determined Mycotoxins in Maize Seedling Blight Caused by Fusarium graminearum

Fusarium graminearum is an important causal agent of maize seedling blight. The species includes several chemotypes that produce various forms of deoxynivalenol (DON) and nivalenol (NIV). To understand the effects and roles of F. graminearum mycotoxins on maize seedling blight occurring at Zhang Ye of Gansu, China, 23 isolates of F. graminearum were collected and characterized. A PCR assay showed all 23 isolates belonged to the 15-acetyldeoxynivalenol (15-ADON) genotype. This was also confirmed by production of both DON and 15-ADON in either rice culture medium or maize seedling roots, detected by high performance liquid chromatography and mass spectrometry. In maize seedling roots, 15-ADON dominated at 6 days post inoculation (dpi) and DON was the main mycotoxin at 12 dpi. The biomass of F. graminearum doubled from 6 to 12 dpi, and was positively correlated with virulence of the isolates. Both mycotoxins affected maize root vitality, but 15-ADON had a greater effect than DON. ALDH9 and MDH, two dehydrogenase synthesis genes in maize, showed a lower relative expression in 15-ADON treatments than in DON treatments. It indicated that both mycotoxins affected seed germination and root development, with 15-ADON being more destructive. Under scanning electron microscopy and transmission electron microscopy, root hair formation and development were delayed by DON, but completely inhibited by 15-ADON. 15-ADON caused cell shrinkage, loose cellular structure, and widened intercellular spaces; it also destroyed organelles and caused plasmolysis, and eventually ruptured cell membranes causing cell death. DON did not affect cell morphology and arrangement, but altered the morphology of organelles, forming concentric membranous bodies and a large amount of irregular lipid droplets. Thus, both mycotoxins contributed to symptom expression of maize seedling blight, but 15-ADON was more destructive than DON.