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Zhang H.,Nanjing Agricultural University | Zhang H.,Key Laboratory of Integrated Management of Crop Diseases and Pests | Zhang H.,State Key Laboratory Breeding Base for Zhejiang Sustainable Plant Pest Control | Zhang H.,MOA Key Laboratory for Pesticide Residue Detection | And 15 more authors.
Journal of Separation Science | Year: 2012

The enantiomeric separation of 21 triazole fungicides was carried out on four polysaccharide-derived chiral stationary phases in the reversed phase separation mode using high performance liquid chromatography coupled with tandem mass spectrometry. All fungicides were detected in electrospray ionization (ESI) positive mode with selected reaction monitoring (SRM). Complete enantioseparation was achieved for 21 fungicides except for difenoconazole based on cellulose tris (3,5-dimethylphenylcarbamate) and cellulose tris (3-chloro-4-methylphenyl carbamate) columns by optimizing experimental conditions including mobile phase and column temperature. Mobile phase was 0.1% formic acid aqueous solution mixed with methanol or acetonitrile in different proportions. Among all the fungicides, 15 with two enantiomers and three with four stereoisomers (bitertanol, bromuconazole, and cyproconazole) were successfully separated at 25°C. Enantioseparation for the other three fungicides (propiconazole, triadimenol, and difenoconazole) with four stereoisomers could be achieved by changing the column temperature from 10 to 40°C. Propiconazole and triadimenol were enantioseparated on baseline at 40 and at 35°C, respectively, and difenoconazole was enantioseparated partially with the Rs > 1.1 at 25°C. Moreover, linearities and limits of detection (LODs) of 21 fungicides except for difenoconazole were studied, showing coefficients of determination (R2) higher than 0.99 and LODs lower than 2.5 μg/L. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hua X.,Nanjing Agricultural University | Hua X.,Key Laboratory of Integrated Management of Crop Diseases and Pests | Yang J.,Henan Academy of Agriculture Science | Wang L.,Nanjing Agricultural University | And 6 more authors.
PLoS ONE | Year: 2012

Objective: Organophosphorus (OP) pesticides are considered hazardous substances because of their high toxicity to nontarget species and their persistence in the environment and agricultural products. Therefore, it is important to develop a rapid, sensitive, and economical method for detecting OP pesticides and their residues in food and the environment. Methods: A broad, selective monoclonal antibody (MAb) for organophosphorus pesticides was produced. Based on the MAb, an enzyme linked immunosorbent assay (ELISA) and an immunochromatography assay (ICA) for detecting OP pesticides in different agricultural products were developed using a binding inhibition format on microtiter plates and a membrane strip, respectively. Results: Under the optimized conditions, the IC50 values of the ELISA ranged from 3.7 to 162.2 ng mL-1 for the 8 OP pesticides. The matrix interferences of Apple, Chinese cabbage, and greengrocery were removed by 40-fold dilution, the recoveries from spiked samples ranged from 79.1% to 118.1%. The IC50 values of ICA for the 8 OP pesticides ranged from 11.8 to 470.4 ng mL-1. The matrix interference was removed from the Chinese cabbage and Apple samples with 5-fold dilution, and the interference was removed from the greengrocery samples with 20-fold dilution. The recoveries from the spiked samples ranged between 70.6 and 131.9%. The established ELISA and ICA were specific selectivity for the 8 OP pesticides. Conclusions: The established ELISA is a sensitive screening method for the detection of OP pesticides, but the ELISA detection method depends on a laboratory platform and requires a relative long assay time and several steps operation. The established ICA is very useful as a screening method for the quantitative, semi-quantitative or qualitative detection of OP pesticides in agricultural products, and it has advantages over ELISA methods with regard to factors such as the testing procedure, testing time, and matrix interferences, among others. © 2012 Hua et al.

Wang J.,Nanjing Agricultural University | Wang J.,Key Laboratory of Integrated Management of Crop Diseases and Pests | Du Y.,Nanjing Agricultural University | Du Y.,Key Laboratory of Integrated Management of Crop Diseases and Pests | And 11 more authors.
Molecular Plant Pathology | Year: 2013

Endocytosis is an essential cellular process in eukaryotic cells that involves concordant functions of clathrin and adaptor proteins, various protein and lipid kinases, phosphatases and the actin cytoskeleton. In Saccharomyces cerevisiae, Ark1p is a member of the serine/threonine protein kinase (SPK) family that affects profoundly the organization of the cortical actin cytoskeleton. To study the function of MoArk1, an Ark1p homologue identified in Magnaporthe oryzae, we disrupted the MoARK1 gene and characterized the ΔMoark1 mutant strain. The ΔMoark1 mutant exhibited various defects ranging from mycelial growth and conidial formation to appressorium-mediated host infection. The ΔMoark1 mutant also exhibited decreased appressorium turgor pressure and attenuated virulence on rice and barley. In addition, the ΔMoark1 mutant displayed defects in endocytosis and formation of the Spitzenkörper, and was hyposensitive to exogenous oxidative stress. Moreover, a MoArk1-green fluorescent protein (MoArk1-GFP) fusion protein showed an actin-like localization pattern by localizing to the apical regions of hyphae. This pattern of localization appeared to be regulated by the N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins MoSec22 and MoVam7. Finally, detailed analysis revealed that the proline-rich region within the MoArk1 serine/threonine kinase (S_TKc) domain was critical for endocytosis, subcellular localization and pathogenicity. These results collectively suggest that MoArk1 exhibits conserved functions in endocytosis and actin cytoskeleton organization, which may underlie growth, cell wall integrity and virulence of the fungus. © 2013 BSPP AND JOHN WILEY & SONS LTD.

Dong Y.,Nanjing Agricultural University | Dong Y.,Key Laboratory of Integrated Management of Crop Diseases and Pests | Li Y.,Nanjing Agricultural University | Li Y.,Key Laboratory of Integrated Management of Crop Diseases and Pests | And 26 more authors.
PLoS Pathogens | Year: 2015

Genome dynamics of pathogenic organisms are driven by pathogen and host co-evolution, in which pathogen genomes are shaped to overcome stresses imposed by hosts with various genetic backgrounds through generation of a variety of isolates. This same principle applies to the rice blast pathogen Magnaporthe oryzae and the rice host; however, genetic variations among different isolates of M. oryzae remain largely unknown, particularly at genome and transcriptome levels. Here, we applied genomic and transcriptomic analytical tools to investigate M. oryzae isolate 98-06 that is the most aggressive in infection of susceptible rice cultivars. A unique 1.4 Mb of genomic sequences was found in isolate 98-06 in comparison to reference strain 70-15. Genome-wide expression profiling revealed the presence of two critical expression patterns of M. oryzae based on 64 known pathogenicity-related (PaR) genes. In addition, 134 candidate effectors with various segregation patterns were identified. Five tested proteins could suppress BAX-mediated programmed cell death in Nicotiana benthamiana leaves. Characterization of isolate-specific effector candidates Iug6 and Iug9 and PaR candidate Iug18 revealed that they have a role in fungal propagation and pathogenicity. Moreover, Iug6 and Iug9 are located exclusively in the biotrophic interfacial complex (BIC) and their overexpression leads to suppression of defense-related gene expression in rice, suggesting that they might participate in biotrophy by inhibiting the SA and ET pathways within the host. Thus, our studies identify novel effector and PaR proteins involved in pathogenicity of the highly aggressive M. oryzae field isolate 98-06, and reveal molecular and genomic dynamics in the evolution of M. oryzae and rice host interactions. © 2015 Dong et al.

Lu C.,Nanjing Agricultural University | Lu C.,Key Laboratory of Integrated Management of Crop Diseases and Pests | Dai T.,Nanjing Agricultural University | Dai T.,Key Laboratory of Integrated Management of Crop Diseases and Pests | And 6 more authors.
Journal of Phytopathology | Year: 2015

We report the development of a loop-mediated isothermal amplification (LAMP) assay targeting the CYP51C element for visual detection of F. oxysporum which caused Fusarium wilt in soybean. The CYP51C-LAMP assay efficiently amplified the target gene in 60 min at 62°C. And specificity was evaluated against F. oxysporum, Fusarium spp. and other fungal species. The detection limit of the CYP51C-specific LAMP assay for F. oxysporum was four conidia per gram soil. The assay also detected F. oxysporum from inoculated soybean tissues and residues. These results suggest that this CYP51C-LAMP assay can be used to detect residues on plants in the field. © 2014 Blackwell Verlag GmbH.

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