Electrical penetration graph technique as a tool to monitor the early stages of aphid resistance to insecticides.

BACKGROUND: Sulfoxaflor, a new insecticide from the sulfoximine chemical family, and imidacloprid, a widely used neonicotinoid insecticide, were tested to assess the susceptibility and feeding behaviour of two populations of Myzus persicae: Mp61, which exhibited target-site R81T resistance to neonicotinoids, and Mp1989, a laboratory clone maintained since 1989 as a susceptible reference. RESULTS: The imidacloprid LC50 value for Mp61 was 16 times higher than for Mp1989, showing a moderate level of resistance. Sulfoxaflor LC50 values for Mp61 and Mp1989 were much closer. The probing behaviour, as assessed by electrical penetration graphs (EPGs), of both populations was clearly altered by sulfoxaflor, which reduced the ability of aphids to find and feed from the phloem. The feeding behaviour of the susceptible Mp1989 population was much more severely affected than the moderately resistant Mp61 population on imidacloprid-treated plants. PCR assays of both aphid populations followed by DNA sequencing identified differences between populations in the point mutation in the ß-subunit of the nicotinic acetylcholine receptor linked to the resistant gene against the neonicotinoid insecticide. CONCLUSIONS: Sulfoxaflor provoked feeding cessation more rapidly than imidacloprid in both aphid populations. Sharp differences in feeding behaviour were detected between the susceptible and the moderately resistant neonicotinoid-resistant aphid populations. The EPG technique can be used as a useful tool to give new insights into the functional effects of new chemical compounds and for early detection of low to moderate levels of resistance of sap-feeding insects to insecticides. The potential of this technique was validated by molecular analysis of the R81T mutation target site.

Epigenetic characterization of the vegetative and floral stages of azalea buds: dynamics of DNA methylation and histone H4 acetylation.

Floral induction in plants is achieved through a complex genetic network and regulated by multiple environmental and endogenous cues. Epigenetic control is determinative in plants for coordinating the switch to flowering under favorable environmental conditions and achieving reproductive success. Global DNA methylation, whose increase is associated with heterochromatinization-cell differentiation, and histone H4 acetylation, which is linked to euchromatin, were analyzed in vegetative and floral buds of azalea in order to study the involvement of epigenetic mechanisms in the floral development of woody plants. The results showed an increase of DNA methylation in floral buds in contrast to the decrease observed for acetylated H4 (AcH4) levels. In addition, when the distributions of 5-mdC and AcH4 in vegetative and floral buds of azalea were analyzed by immunodetection, opposite patterns in their distribution were revealed and confirmed the existence of different cell types in the shoot apical meristem with varying degrees of differentiation.

HPCE quantification of 5-methyl-2'-deoxycytidine in genomic DNA: methodological optimization for chestnut and other woody species.

Quantification of deoxynucleosides using micellar high-performance capillary electrophoresis (HPCE) is an efficient, fast and inexpensive evaluation method of genomic DNA methylation. This approach has been demonstrated to be more sensitive and specific than other methods for the quantification of DNA methylation content. However, effective detection and quantification of 5-methyl-2'-deoxycytidine depend of the sample characteristics. Previous works have revealed that in most woody species, the quality and quantity of RNA-free DNA extracted that is suitable for analysis by means of HPCE varies among species of the same gender, among tissues taken from the same tree, and vary in the same tissue depending on the different seasons of the year. The aim of this work is to establish a quantification method of genomic DNA methylation that lends itself to use in different Castanea sativa Mill. materials, and in other angiosperm and gymnosperm woody species. Using a DNA extraction kit based in silica membrane has increased the resolutive capacity of the method. Under these conditions, it can be analyzed different organs or tissues of angiosperms and gymnosperms, regardless of their state of development. We emphasized the importance of samples free of nucleosides, although, in the contrary case, the method ensures the effective separation of deoxynucleosides and identification of 5-methyl-2'-deoxycytidine.