Molecular evidence of Cacao swollen shoot virus acquisition and retention by Planococcus citri (Risso) and Pseudococcus longispinus (Targioni-Tozzetti) and Pseudococcus viburni (Signoret) Mealybugs (Hemiptera: Pseudococcidae)

Cacao swollen shoot virus (CSSV) remains a significant viral pathogen endemic in West African cacao-growing countries. Eradication of affected trees (complete removal) and resistance breeding in existing cacao (Theobroma cacao L.) germplasm have not yielded the expected results in terms of controlling the spread of CSSV. CSSV is reported to be vectored by at least 16 species of mealybugs (Hemiptera: Pseudococcidae), but there have been no molecular evidences to validate these reports. Inconsistent reports exit on CSSV transmission which were based on visual assessments of CSSV infection which are highly subjective, especially on asymptomatic cacao. The validation of the transmission efficiency of CSSV by mealybugs are arguable. In this study, the presence of CSSV in three mealybug species, Planococcus citri (Risso), Pseudococcus longispinus (Targioni Tozzetti) and Pseudococcus viburni (Signoret), was monitored via PCR after a 72-h virus acquisition access period (AAP) on symptomatic T. cacao (var. Amelonado) seedlings ‘infected’ with New Juaben, a severe strain of CSSV. Solanum tuberosum sprouts were then fed to the mealybugs over a sixday period. Sequenced PCR products of the destructively sampled and CSSV-screened individual mealybug species provided a molecular evidence of CSSV acquisition. qPCR results gave another evidence that CSSV retention is highly variable between and within mealybug species. This is the first molecular evidence highlighting a non-West African mealybug species, Ps. viburni, as a capable CSSV vector. Apparently, these results could validate the efficacy of non-malvaceous species as barrier crops and in screening for resistance to CSSV vectors in T. cacao

Spontaneous gene flow from rapeseed (Brassica napus) to wild Brassica oleracea

Research on the environmental risks of gene flow from genetically modified (GM) crops to wild relatives has traditionally emphasized recipients yielding most hybrids. For GM rapeseed (Brassica napus), interest has centred on the ‘frequently hybridizing’ Brassica rapa over relatives such as Brassica oleracea, where spontaneous hybrids are unreported in the wild. In two sites, where rapeseed and wild B. oleracea grow together, we used flow cytometry and crop-specific microsatellite markers to identify one triploid F(1) hybrid, together with nine diploid and two near triploid introgressants. Given the newly discovered capacity for spontaneous introgression into B. oleracea, we then surveyed associated flora and fauna to evaluate the capacity of both recipients to harm cohabitant species with acknowledged conservational importance. Only B. oleracea occupies rich communities containing species afforded legislative protection; these include one rare micromoth species that feeds on B. oleracea and warrants further assessment. We conclude that increased attention should now focus on B. oleracea and similar species that yield few crop-hybrids, but possess scope to affect rare or endangered associates

Brassica and Sinapis Seeds in Medieval Archaeological Sites:An Example of Multiproxy Analysis for Their Identification and Ethnobotanical Interpretation

The genus Brassica includes some of the most important vegetable and oil crops worldwide. Many Brassica seeds (which can show diagnostic characters useful for species identification) were recovered from two archaeological sites in northern Italy, dated from between the Middle Ages and the Renaissance. We tested the combined use of archaeobotanical keys, ancient DNA barcoding, and references to ancient herbarium specimens to address the issue of diagnostic uncertainty. An unequivocal conventional diagnosis was possible for much of the material recovered, with the samples dominated by five Brassica species and Sinapis. The analysis using ancient DNA was restricted to the seeds with a Brassica-type structure and deployed a variant of multiplexed tandem PCR. The quality of diagnosis strongly depended on the molecular locus used. Nevertheless, many seeds were diagnosed down to species level, in concordance with their morphological identification, using one primer set from the core barcode site (matK). The number of specimens found in the Renaissance herbaria was not high; Brassica nigra, which is of great ethnobotanical importance, was the most common taxon. Thus, the combined use of independent means of species identification is particularly important when studying the early use of closely related crops, such as Brassicaceae

Using DNA Metabarcoding to Identify the Floral Composition of Honey:A New Tool for Investigating Honey Bee Foraging Preferences

Identifying the floral composition of honey provides a method for investigating the plants that honey bees visit. We compared melissopalynology, where pollen grains retrieved from honey are identified morphologically, with a DNA metabarcoding approach using the rbcL DNA barcode marker and 454-pyrosequencing. We compared nine honeys supplied by beekeepers in the UK. DNA metabarcoding and melissopalynology were able to detect the most abundant floral components of honey. There was 92% correspondence for the plant taxa that had an abundance of over 20%. However, the level of similarity when all taxa were compared was lower, ranging from 22–45%, and there was little correspondence between the relative abundance of taxa found using the two techniques. DNA metabarcoding provided much greater repeatability, with a 64% taxa match compared to 28% with melissopalynology. DNA metabarcoding has the advantage over melissopalynology in that it does not require a high level of taxonomic expertise, a greater sample size can be screened and it provides greater resolution for some plant families. However, it does not provide a quantitative approach and pollen present in low levels are less likely to be detected. We investigated the plants that were frequently used by honey bees by examining the results obtained from both techniques. Plants with a broad taxonomic range were detected, covering 46 families and 25 orders, but a relatively small number of plants were consistently seen across multiple honey samples. Frequently found herbaceous species were Rubus fruticosus, Filipendula ulmaria, Taraxacum officinale, Trifolium spp., Brassica spp. and the non-native, invasive, Impatiens glandulifera. Tree pollen was frequently seen belonging to Castanea sativa, Crataegus monogyna and species of Malus, Salix and Quercus. We conclude that although honey bees are considered to be supergeneralists in their foraging choices, there are certain key species or plant groups that are particularly important in the honey bees environment. The reasons for this require further investigation in order to better understand honey bee nutritional requirements. DNA metabarcoding can be easily and widely used to investigate floral visitation in honey bees and can be adapted for use with other insects. It provides a starting point for investigating how we can better provide for the insects that we rely upon for pollination

DNA barcoding the native flowering plants and conifers of Wales

We present the first national DNA barcode resource that covers the native flowering plants and conifers for the nation of Wales (1143 species). Using the plant DNA barcode markers rbcL and matK, we have assembled 97.7% coverage for rbcL, 90.2% for matK, and a dual-locus barcode for 89.7% of the native Welsh flora. We have sampled multiple individuals for each species, resulting in 3304 rbcL and 2419 matK sequences. The majority of our samples (85%) are from DNA extracted from herbarium specimens. Recoverability of DNA barcodes is lower using herbarium specimens, compared to freshly collected material, mostly due to lower amplification success, but this is balanced by the increased efficiency of sampling species that have already been collected, identified, and verified by taxonomic experts. The effectiveness of the DNA barcodes for identification (level of discrimination) is assessed using four approaches: the presence of a barcode gap (using pairwise and multiple alignments), formation of monophyletic groups using Neighbour-Joining trees, and sequence similarity in BLASTn searches. These approaches yield similar results, providing relative discrimination levels of 69.4 to 74.9% of all species and 98.6 to 99.8% of genera using both markers. Species discrimination can be further improved using spatially explicit sampling. Mean species discrimination using barcode gap analysis (with a multiple alignment) is 81.6% within 10×10 km squares and 93.3% for 2×2 km squares. Our database of DNA barcodes for Welsh native flowering plants and conifers represents the most complete coverage of any national flora, and offers a valuable platform for a wide range of applications that require accurate species identification

Residual ground-water levels of the neonicotinoid thiacloprid perturb chemosensing of Caenorhabditis elegans

© 2017, The Author(s). This study investigated the neurological effects of residual ground-water levels of thiaclopridon the non-target organism Caenorhabditis elegans. Nematodes treated with thiacloprid showed a dose-dependent and significantly increased twitch response at concentrations above 50 ng mL−1 that disabled their forward locomotion in liquid culture. In comparison with untreated controls, 10 ng mL−1 thiacloprid perturbed the chemosensory ability of C. elegans such that the nematodes no longer demonstrated positive chemotaxis towards a NaCl chemo-attractant, reducing their chemotaxis index from +0.48 to near to zero. Nematodes also exhibited a locomotion characteristic of those devoid of chemo-attraction, making significantly more pirouetting turns of ≥90° than the untreated controls. Compared to the untreated controls, expression of the endocytosis-associated gene, Rab-10, was also increased in C. elegans that had developed to adulthood in the presence of 10 ng mL−1 thiacloprid, suggesting their active engagement in increased recycling of affected cellular components, such as their nAChRs. Thus, even residual, low levels of this less potent neonicotinoid that may be found in field ground-water had measurable effects on a beneficial soil organism which may have environmental and ecological implications that are currently poorly understood

Spontaneous capture of oilseed rape (Brassica napus) chloroplasts by wild B. rapa: implications for the use of chloroplast transformation for biocontainment

Environmental concerns over the cultivation of Genetically Modified (GM) crops largely centre on the ecological consequences following gene flow to wild relatives. One attractive solution is to deploy biocontainment measures that prevent hybridization. Chloroplast transformation is the most advanced biocontainment method but is compromised by chloroplast capture (hybridization through the maternal lineage). To date, however, there is a paucity of information on the frequency of chloroplast capture in the wild. Oilseed rape (Brassica napus, AACC) frequently hybridises with wild Brassica rapa (AA, as paternal parent) and yields B. rapa-like introgressed individuals after only two generations. In this study we used chloroplast CAPS markers that differentiate between the two species to survey wild and weedy populations of B. rapa for the capture of B. napus chloroplasts. A total of 464 B. rapa plants belonging to 14 populations growing either in close proximity to B. napus (i.e. sympatric 1 km) were assessed for chloroplast capture using PCR (trnL-F) and CAPS (trnT-L-Xba I) markers. The screen revealed that two sympatric B. rapa populations included 53 plants that possessed the chloroplast of B. napus. In order to discount these B. rapa plants as F1 crop-wild hybrids, we used a C-genome-specific marker and found that 45 out of 53 plants lacked the C-genome and so were at least second generation introgressants. The most plausible explanation is that these individuals represent multiple cases of chloroplast capture following introgressive hybridisation through the female germ line from the crop. The abundance of such plants in sympatric sites thereby questions whether the use of chloroplast transformation would provide a sufficient biocontainment for GM oilseed rape in the United Kingdom.Nadia Haider, Joel Allainguillaume and Mike J. Wilkinso