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Wang H.,Guizhou Tobacco Science Institute | Li W.,Guizhou Institute of Plant Protection | Chen Q.,Guizhou Tobacco Science Institute | Huang Y.,Yangtze University | And 6 more authors.
Phytopathology | Year: 2012

A simple, rapid, small-scale microbioassay for infection of tobacco seedlings by Phytophthora parasitica var. nicotianae was developed here. This assay uses tobacco seedlings cultivated in petri dishes for a standardized method for quantitation of initial zoospore inocula and highthroughput screening of antagonistic bacteria. Zoospore inocula between 102 to 105 spores per petri dish were inoculated on 14-day-old tobacco seedlings for the susceptibility test. The optimum inocula was established to be ten thousand zoospores. One hundred and fifty pure culture bacteria with different pigments, growth rates, and morphologies were isolated from rhizosphere soil of tobacco and screened for protective ability against tobacco black shank. Fifteen bacteria presented high activity against P. parasitica on tobacco seedlings. They were identified by Biolog GEN III MicroPlate and distributed as Bacillus amyloliquefaciens, B. licheniformis, Paenibacillus pabuli, B. atrophaeus, B. subtilis, B. pumilus, and B. endophyticus, respectively. Four antagonists chosen randomly from the 15 bacteria all exhibited the same 100% protective activity in planta as that in the petri dishes. This microassay proved to be a rapid, reproducible, and efficient method for screening of potential biological agents or microorganisms and may be useful for studying mechanisms of infection and control of Phytophthora spp. Under hydroponic conditions. © 2012 The American Phytopathological Society.


Wang H.,Guizhou Tobacco Science Institute | Yang S.,Guizhou Province Tobacco Company | Yang S.,China Agricultural University | Wang M.,Guizhou Tobacco Science Institute | And 8 more authors.
Crop Protection | Year: 2013

Tobacco black shank caused by Phytophthora parasitica is a devastating soil-borne disease of tobacco in China. Control of tobacco black shank relies on numerous fungicide applications. A new carboxylic acid amide (CAA) fungicide, mandipropamid, was examined for its effects on various asexual developmental stages of P. parasitica in vitro and in tobacco seedlings. Although mandipropamid did not affect discharge of zoospores from sporangia, it strongly inhibited mycelial growth (mean EC50 value of 0.0112 μg ml-1), sporangia production (mean EC50 value of 0.009 μg ml-1), germination of encysted zoospores (mean EC50 value of 0.014 μg ml-1), and germination of sporangia (mean EC50 value of 0.017 μg ml-1). For protective activity in tobacco seedlings, various applications of mandipropamid were superior in reducing black shank compared to that of metalaxyl and of azoxystrobin; while for curative activity assay, 100 μg ml-1 of mandipropamid exhibited the same efficacy as that of metalaxyl, and presented superior activity than that of azoxystrobin. In 2010 and 2011, 119 isolates of P. parasitica from Guizhou Province of China were characterized for the baseline sensitivity to mandipropamid by measuring mycelial growth. Values of effective concentrations for 50% mycelia growth inhibition varied from 0.0068 to 0.0285 μg ml-1 and averaged 0.013 (±0.0045) μg ml-1, with a unimodal distribution. This information will serve as a baseline for tracking future changes in sensitivities of P. parasitica populations to mandipropamid in China. © 2012 Elsevier Ltd.


Li H.-B.,Yangzhou University | Li H.-B.,Guizhou Institute of Plant Protection | Zheng Y.-T.,Yangzhou University | Sun D.-D.,Yangzhou University | And 2 more authors.
Pesticide Biochemistry and Physiology | Year: 2014

Temperature and pesticide are two important factors that affect survival, reproduction and other physiological processes of insects. To determine interactions of elevated temperature and avermectins treatment on the western flower thrips, Frankliniella occidentalis, newly emerged adults were exposed to combinations of three temperatures (21, 26 and 33. °C) and two avermectins concentrations (0, 45. ppm), and survival rate, reproduction, longevity, antioxidant enzymes activities and heat shock proteins (hsps) induction were analyzed. The results showed that the survival, longevity and reproduction of F. occidentalis decreased with increased temperature and avermectins treatment. While elevated temperature and avermectins treatment significantly decreased activity of SOD, activities of POD and GST significantly increased after exposure to elevated temperature, avermectins or their combination. Elevated temperature had no effect on activity of CAT, but it was obviously improved by the combination of temperature and avermectins treatment. Expression analysis of hsps showed that four heat shock proteins (hsp90, hsc702, hsp60 and hop) were up-regulated by the induction of elevated temperature with small fold changes. After treatment with avermectins, expression levels of hsp90, hsc701, hsc702 and hop were significantly up-regulated with increased temperature and higher than those of their respective control at higher temperature. Surprisingly, expression level of hps60 was down-regulated with increased temperature, but the expression level at 21 or 26. °C remained higher than that of control. Overall, our studies suggest that elevated temperature enhance toxicity of avermectins and their combination induced acute oxidative damage to F. occidentalis. Therefore, consideration of temperature in evaluating avermectins toxicity is necessary to make accurate prediction of its effect on F. occidentalis and other insects. © 2014 Elsevier Inc.


Zheng Y.-T.,Yangzhou University | Li H.-B.,Yangzhou University | Li H.-B.,Guizhou Institute of Plant Protection | Lu M.-X.,Yangzhou University | Du Y.-Z.,Yangzhou University
PLoS ONE | Year: 2014

Quantitative real time PCR (qRT-PCR) has emerged as a reliable and reproducible technique for studying gene expression analysis. For accurate results, the normalization of data with reference genes is particularly essential. Once the transcriptome sequencing of Frankliniella occidentalis was completed, numerous unigenes were identified and annotated. Unfortunately, there are no studies on the stability of reference genes used in F. occidentalis. In this work, seven candidate reference genes, including actin, 18S rRNA, H3, tubulin, GAPDH, EF-1 and RPL32, were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs BestKeeper, geNorm, Normfinder and the comparative DCt method. Because the rankings of the reference genes provided by each of the four programs were different, we chose a user-friendly web-based comprehensive tool RefFinder to get the final ranking. The result demonstrated that EF-1 and RPL32 displayed the most stable expression in different developmental stages; RPL32 and GAPDH showed the most stable expression at high temperatures, while 18S and EF-1 exhibited the most stable expression at low temperatures. In this study, we validated the suitable reference genes in F. occidentalis for gene expression profiling under different experimental conditions. The choice of internal standard is very important in the normalization of the target gene expression levels, thus validating and selecting the best genes will help improve the quality of gene expression data of F. occidentalis. What is more, these validated reference genes could serve as the basis for the selection of candidate reference genes in other insects. Copyright © 2014 Zheng et al.


Lu M.-X.,Yangzhou University | Li H.-B.,Yangzhou University | Li H.-B.,Guizhou Institute of Plant Protection | Zheng Y.-T.,Yangzhou University | And 2 more authors.
Journal of Thermal Biology | Year: 2016

The western flower thrips, Frankliniella occidentalis, is an important invasive pest with a strong tolerance for extreme temperatures; however, the molecular mechanisms that regulate thermotolerance in this insect remain unclear. In this study, four heat shock protein genes were cloned from F. occidentalis and named Fohsp90, Fohsc701, Fohsc702 and Fohsp60. These four Hsps exhibited typical characteristics of heat shock proteins. Subcellular localization signals and phylogenetic analysis indicated that FoHsp90 and FoHsc701 localize to the cytosol, whereas FoHsc702 and FoHsp60 were located in the endoplasmic reticulum and mitochondria, respectively. Analysis of genomic sequences revealed the presence of introns in the four genes (three, four, seven, and five introns for Fohsp90, Fohsc701, Fohsc702 and Fohsp60, respectively). Both the number and position of introns in these four genes were quite different from analogous genes in other species. qRT-PCR indicated that the four Fohsps were detected in second-stage larvae, one-day-old pupae, and one-day-old adults, and mRNA expression levels were lowest in larvae and highest in pupae. Fohsc701 and Fohsc702 possessed similar expression patterns and were not induced by cold or heat stress. Expression of Fohsp60 was significantly elevated by heat, and Fohsp90 was rapidly up-regulated after exposure to both cold and heat stress. Exposure to -8 °C had no effect on expression of the four Fohsps; however, expression of Fohsp90 and Fohsp60 was highest after a 2-h incubation at 39 °C. Furthermore, cold and heat hardening led to significant up-regulation of the four Fohsps compared to their respective controls. Collectively, our results indicate that the four FoHsps contribute to insect development and also function in rapid cold or heat hardening; furthermore, FoHsp90 and FoHsp60 contribute to thermotolerance in F. occidentalis. © 2016 Elsevier Ltd.

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