135 research outputs found

    Development of a sensitive nested-polymerase chain reaction (PCR) assay for the detection of Ustilago scitaminea

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    A species-specific polymerase chain reaction (PCR) assay was developed for rapid and accurate detection of Ustilago scitaminea, the causal agent of sugarcane smut disease. Based on nucleotide differences in the internal transcribed spacer (ITS) sequences of U. scitaminea, a pair of species-specific primers, SL1 (5`-CAGTGCACGAAAGTACCTGTGG-3`) and SR2 (5`-CTAGGGCGGTGTTCAGAAGCAC-3`) was designed by using a panel of fungal and bacterial species as controls. The primers SL1/SR2 specifically amplified a unique PCR product about 530 bp in length from U. scitaminea strains with a detecting sensitivity at 200 fg of the fungal genomic DNA in a 25 μl reaction solution. To increase sensitivity, a nested-PCR protocol was further established, which used ITS4/ITS5 as the first-round primers followed by the primer pair SL1/SR2. This protocol increased the detection sensitivity by 10,000-fold compared to the PCR method and could detect the fungal DNA as low as 20 ag. The nested-PCR detected U. scitaminea from young sugarcane leaves with no visible smut disease symptoms. The findings from this study provide a sensitive and reliable technique for the early detection of U. scitaminea, which would be useful for sugarcane quarantine and production of germ-free seedcanes.Keywords: Sugarcane, Ustilago scitaminea, nested-polymerase chain reaction (PCR), molecular detectio

    Characterization of a broad-based mosquito yeast interfering RNA larvicide with a conserved target site in mosquito semaphorin-1a genes

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    BACKGROUND: RNA interference (RNAi), which has facilitated functional characterization of mosquito neural development genes such as the axon guidance regulator semaphorin-1a (sema1a), could one day be applied as a new means of vector control. Saccharomyces cerevisiae (baker's yeast) may represent an effective interfering RNA expression system that could be used directly for delivery of RNA pesticides to mosquito larvae. Here we describe characterization of a yeast larvicide developed through bioengineering of S. cerevisiae to express a short hairpin RNA (shRNA) targeting a conserved site in mosquito sema1a genes. RESULTS: Experiments conducted on Aedes aegypti larvae demonstrated that the yeast larvicide effectively silences sema1a expression, generates severe neural defects, and induces high levels of larval mortality in laboratory, simulated-field, and semi-field experiments. The larvicide was also found to induce high levels of Aedes albopictus, Anopheles gambiae and Culex quinquefasciatus mortality. CONCLUSIONS: The results of these studies indicate that use of yeast interfering RNA larvicides targeting mosquito sema1a genes may represent a new biorational tool for mosquito control

    Genetic diversity of Ustilago scitaminea Syd. in Southern China revealed by combined ISSR and RAPD analysis

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    The polymorphism and similarity relationships among 35 mating-type isolates of Ustilago scitaminea collected from Southern China were determined with random amplified polymorphic DNA (RAPD) and inter-simple  sequence repeat (ISSR) analyses. These fungal isolates were collected from 16 sugarcane cultivars including F134 that is resistant to the physiological race 1 but susceptible to the race 2 of U. scitaminea, and N: Co376  that is immune to both races 1 and 2. Unweighted pair group method with arithmetic mean (UPGMA) cluster  analysis revealed that the U. scitaminea isolates could be divided into 2 groups with a coefficient of 0.74. The  first group comprises two isolates collected from the sugarcane cultivar F134, while the remaining 33 isolates were clustered into the second group. The second group was further divided into two subgroups with most of  the isolates from Guangdong Province which clustered in the same subgroup, and all the isolates from Guangxi  and Yunnan Provinces were clustered in another subgroup. Given that the member of the second group could  infect the cultivar N:Co376, which is immune to the races 1 and 2, our results suggest that majority of U.  scitaminea in sugarcane-producing regions of Southern China may belong to or genetically similar to race 3.Key words: Ustilago scitaminea, sugarcane, inter-simple sequence repeat (ISSR), random amplified  polymorphic deoxyribonucleic acid (DNA) (RAPD), genetic diversity

    Cross-Layer Adaptive Feedback Scheduling of Wireless Control Systems

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    There is a trend towards using wireless technologies in networked control systems. However, the adverse properties of the radio channels make it difficult to design and implement control systems in wireless environments. To attack the uncertainty in available communication resources in wireless control systems closed over WLAN, a cross-layer adaptive feedback scheduling (CLAFS) scheme is developed, which takes advantage of the co-design of control and wireless communications. By exploiting cross-layer design, CLAFS adjusts the sampling periods of control systems at the application layer based on information about deadline miss ratio and transmission rate from the physical layer. Within the framework of feedback scheduling, the control performance is maximized through controlling the deadline miss ratio. Key design parameters of the feedback scheduler are adapted to dynamic changes in the channel condition. An event-driven invocation mechanism for the feedback scheduler is also developed. Simulation results show that the proposed approach is efficient in dealing with channel capacity variations and noise interference, thus providing an enabling technology for control over WLAN.Comment: 17 pages, 12 figures; Open Access at http://www.mdpi.org/sensors/papers/s8074265.pd

    Examination of the genetic basis for sexual dimorphism in the Aedes aegypti (dengue vector mosquito) pupal brain

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    BACKGROUND: Most animal species exhibit sexually dimorphic behaviors, many of which are linked to reproduction. A number of these behaviors, including blood feeding in female mosquitoes, contribute to the global spread of vector-borne illnesses. However, knowledge concerning the genetic basis of sexually dimorphic traits is limited in any organism, including mosquitoes, especially with respect to differences in the developing nervous system. METHODS: Custom microarrays were used to examine global differences in female vs. male gene expression in the developing pupal head of the dengue vector mosquito, Aedes aegypti. The spatial expression patterns of a subset of differentially expressed transcripts were examined in the developing female vs. male pupal brain through in situ hybridization experiments. Small interfering RNA (siRNA)-mediated knockdown studies were used to assess the putative role of Doublesex, a terminal component of the sex determination pathway, in the regulation of sex-specific gene expression observed in the developing pupal brain. RESULTS: Transcripts (2,527), many of which were linked to proteolysis, the proteasome, metabolism, catabolic, and biosynthetic processes, ion transport, cell growth, and proliferation, were found to be differentially expressed in A. aegypti female vs. male pupal heads. Analysis of the spatial expression patterns for a subset of dimorphically expressed genes in the pupal brain validated the data set and also facilitated the identification of brain regions with dimorphic gene expression. In many cases, dimorphic gene expression localized to the optic lobe. Sex-specific differences in gene expression were also detected in the antennal lobe and mushroom body. siRNA-mediated gene targeting experiments demonstrated that Doublesex, a transcription factor with consensus binding sites located adjacent to many dimorphically expressed transcripts that function in neural development, is required for regulation of sex-specific gene expression in the developing A. aegypti brain. CONCLUSIONS: These studies revealed sex-specific gene expression profiles in the developing A. aegypti pupal head and identified Doublesex as a key regulator of sexually dimorphic gene expression during mosquito neural development

    Requirement for commissureless2 function during dipteran insect nerve cord development

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    BACKGROUND: In Drosophila melanogaster, commissureless (comm) function is required for proper nerve cord development. Although comm orthologs have not been identified outside of Drosophila species, some insects possess orthologs of Drosophila comm2, which may also regulate embryonic nerve cord development. Here, this hypothesis is explored through characterization of comm2 genes in two disease vector mosquitoes. RESULTS: Culex quinquefasciatus (West Nile and lymphatic filiariasis vector) has three comm2 genes that are expressed in the developing nerve cord. Aedes aegypti (dengue and yellow fever vector) has a single comm2 gene that is expressed in commissural neurons projecting axons toward the midline. Loss of comm2 function in both A. aegypti and D. melanogaster was found to result in loss of commissure defects that phenocopy the frazzled (fra) loss of function phenotypes observed in both species. Loss of fra function in either insect was found to result in decreased comm2 transcript levels during nerve cord development. CONCLUSIONS: The results of this investigation suggest that Fra down-regulates repulsion in precrossing commissural axons by regulating comm2 levels in both A. aegypti and D. melanogaster, both of which require Comm2 function for proper nerve cord development
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