A next generation sequencing approach to elucidate CSSV species profiles

Cacao swollen shoot virus (CSSV) is a member of the family Caulimoviridae, genus Badnavirus and is naturally transmitted to Theobroma cacao by several mealybug species. The virus is restricted to West Africa, while the cacao tree originates from the Americas, and has therefore most probably an indigenous origin on the West African subcontinent. The resultant disease has caused enormous economic damage in Ghana since the 1930's but was restricted to small areas in Togo and Côte d'Ivoire until recently. Now, renewed outbreaks in the main producing areas in Côte d'Ivoire, Ghana and Togo, cause serious yield losses and tree death. CSSV populations in West African countries are genetically structured into several different groups according to the diversity in the first part of ORF3 corresponding to the movement protein. To unravel the extent of isolate diversity we used Illumina HiSeq technology and reconstructed 21 new complete genomes corresponding to the different groups of CSSV sequences. In this way we were able to compare the partial sequences of the RTase region (recognised as the taxonomical region by ICTV using a 20% threshold of nucleotide divergence to denote separate species), and thereby identifying nine different CSSV species. These results will now be used to improve the detection of all badnaviruses present in cacao leaf samples, a vital tool in efforts to halt the spread of the disease and confirm the healthy status of new plantations. (Résumé d'auteur

Electropenetrography application and molecular-based virus detection in mealybug (Hemiptera: Pseudococcidae) vectors of Cacao swollen shoot virus on Theobroma cacao L.

© 2018 Cacao swollen shoot virus (CSSV) is a peril exclusive to the West African cacao-growing countries; causing the Cacao swollen shoot virus disease. This study was set out (1) to analyse the feeding behaviour of two West African and one non-West African mealybug species, Planococcus citri (Risso) and Pseudococcus longispinus (Targioni Tozzetti) and Ps. viburni (Signoret) respectively on CSSV-free cacao. and (2) to provide molecular-based information on the ability of these mealybugs to acquire and transmit the ‘New Juaben’ CSSV strain from CSSV-infected cacao. Electrical penetration graph (EPG) analysis established that these three mealybug species performed both extracellular (C, E1e, F, G and Np waveforms) and intracellular (E1 and E2 waveforms) feeding activities on cacao which were typical of stylet-possessing, phloem-feeding, virus transmitting hemipterans. Waveform F reported in this study is the first for Pl. citri, Ps. longispinus and Ps. viburni feeding on cacao. The competitive feeding efficiency of Ps. viburni on cacao highlights its potential as a ‘new’ vector of CSSV. PCR-based results show that Pl. citri, Ps. longispinus and Ps. viburni can acquire CSSV after a 72-h access acquisition period (AAP). DNA sequences of CSSV were detected in leaf tissues of the test plants after a 30-day post 72-h inoculation access period (IAP) by the viruliferous mealybug individuals. It is the first report, with molecular evidence, of T. cacao serving as an acceptable host to Ps. viburni

Rapid identification of bacteria associated with Acute Oak Decline by high-resolution melt analysis

© 2016 The Society for Applied Microbiology Two Gram-negative Enterobacteriaceae, Gibbsiella quercinecans and Brenneria goodwinii, are frequently isolated from oak suffering from Acute Oak Decline. These two species are difficult to identify based on colony morphology, carbohydrate utilization or 16S rRNA gene sequence, and identification using gyrB gene sequencing is time-consuming and laborious. A rapid identification technique, based on high-resolution melt analysis of the atpD gene, was designed to efficiently process numerous isolates from an increasing number of affected woodlands and parks. Principal component analysis of the resulting melt curves from strains of G.quercinecans, B.goodwinii and their close phylogenetic relatives allowed differentiation into distinct clusters based on species or subspecies identity. Significance and Impact of the Study: Acute Oak Decline is an increasing threat to Britain's native oak population. Two novel bacterial species both belonging to the family Enterobacteriaceae, Gibbsiella quercinecans and Brenneria goodwinii, are thought to play an important role in symptom development. Here, we describe a rapid identification technique using high-resolution melt analysis of the atpD gene able to assign isolates to either G.quercinecans or B.goodwinii in a single assay, greatly reducing the time taken to identify if either or both of these species are present in symptomatic oak

Pseudomonas kirkiae sp. nov., a novel species isolated from oak in the United Kingdom, and phylogenetic considerations of the genera Pseudomonas, Azotobacter and Azomonas

As the current episode of Acute Oak Decline (AOD) continues to affect native British oak in the United Kingdom, ongoing isola-tions from symptomatic and healthy oak have yielded a large Pseudomonas species population. These strains could be divided into taxa representing three potential novel species. Recently, two of these taxa were described as novel Pseudomonas species in the Pseudomonas fluorescens lineage. Here, we demonstrate using a polyphasic approach that the third taxon represents another novel Pseudomonas species. The 16S rRNA gene sequencing assigned the strains to the Pseudomonas aeruginosa lineage, while multilocus sequence analysis (based on partial gyrB, rpoB and rpoD sequences) placed the 13 strains in a single cluster on the border of the Pseudomonas stutzeri group. Whole genome intra-species comparisons (based on average nucleotide identity and in silico DNA–DNA hybridization) confirmed that the strains belong to a single taxon, while the inter-species comparisons with closest phylogenetic relatives yielded similarity values below the accepted species threshold. Therefore, we propose these strains as a novel species, namely Pseudomonas kirkiae sp. nov., with the type strain FRB 229T (P4CT=LMG 31089T=NCPPB 4674T). The phylogenetic analyses performed in this study highlighted the difficulties in assigning novel species to the genus Pseudomonas due to its polyphyletic nature and close relationship to the genus Azotobacter. We further propose that a thorough taxonomic re-evaluation of the genus Pseudomonas is essential and should be performed in the near future

Identification and distribution of novel badnaviral sequences integrated in the genome of cacao (Theobroma cacao)

As part of an ongoing study to understand the diversity of the badnavirus complex, responsible for the cacao swollen shoot disease in West Africa, evidence was found recently of virus-like sequences in asymptomatic cacao plants. The present study exploited the wealth of genomic resources in this crop, and combined bioinformatic, molecular, and genetic approaches to report for the first time the presence of integrated badnaviral sequences in most of the cacao genetic groups. These sequences, which we propose to name eTcBV for endogenous Theobroma cacao bacilliform viruses, varied in type with each predominating in a specific cacao genetic group. Additionally to the viral insert of type VI first identified, we recently described, with the help of Oxford Nanopore technology, viral inserts of type I, II, III and V longer than 10kb. A diagnostic multiplex PCR method was developed to identify the homozygous or hemizygous condition of the specific insert of type VI, which was inherited as a single Mendelian trait. These data suggest that these integration events occurred before or during the species diversification in Central and South America, and prior to its cultivation in other regions. Such evidence of integrated sequences is relevant to the management of cacao quarantine facilities, and may also aid novel methods to reduce the impact of such viruses in this crop

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

Statement on a conceptual framework for the risk assessment of certain food additives re-evaluated under Commission Regulation (EU) No 257/2010

The Panel on Food Additives and Nutrient Sources added to Food (ANS) provides a scientific statement presenting a conceptual framework for the risk assessment of certain food additives re-evaluated under Commission Regulation (EU) No 257/2010. This framework will be used in the evaluation made by the Panel, but the expert judgement of the scientific background, on a case-by-case basis, remains essential to reach a final conclusion. The outcome of the re-evaluation of food additives taking into account all available information is presented in the document, as well as the exposure assessment scenarios to be carried out by the Panel considering the use levels set in the legislation and the availability of adequate usage or analytical data

Using DNA metabarcoding to identify the floral composition of honey: A new tool for investigating honey bee foraging preferences

Copyright © 2015 Hawkins et al. 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

Pseudomonas daroniae sp. nov. and Pseudomonas dryadis sp. nov., isolated from pedunculate oak affected by acute oak decline in the UK

Twenty-two cream-coloured bacterial strains were isolated from oak trees affected by acute oak decline (AOD) in Southern England. Isolates were Gram-negative, motile, slightly curved rods, aerobic, non-spore-forming, catalase positive and oxidase positive. 16S rRNA gene sequence analysis placed the strains in two separate phylogenetic clusters in the Pseudomonas straminea group, with Pseudomonas flavescens as the closest phylogenetic relative. Multilocus sequence analyses of the gyrB, rpoD and rpoB genes supported the delineation of the strains into two separate taxa, which could be differentiated phenotypically and chemotaxonomically from each other, and their closest relatives. Average nucleotide identity and in silico DNA-DNA hybridization values revealed percentages of genome similarity below the species threshold (95 and 70 %, respectively) between the two taxa and the closest relatives, confirming their novel species status. Therefore, on the basis of this polyphasic approach we propose two novel Pseudomonas species, Pseudomonasdaroniae sp. nov. (type strain FRB 228T=LMG 31087T=NCPPB 4672T) and Pseudomonasdryadis sp. nov. (type strain FRB 230T=LMG 31087T=NCPPB 4673T)

Lemurs in Cacao: Presence and Abundance within the Shade Plantations of Northern Madagascar

© 2019 S. Karger AG, Basel. The recognition that much biodiversity exists outside protected areas is driving research to understand how animals survive in anthropogenic landscapes. In Madagascar, cacao (Theobroma cacao) is grown under a mix of native and exotic shade trees, and this study sought to understand whether lemurs were present in these agroecosystems. Between November 2016 and March 2017, discussions with farmers, nocturnal reconnaissance surveys and camera traps were used to confirm the presence of lemurs in the Cokafa and Mangabe plantations near Ambanja, north-west Madagascar. Four species of lemur were encountered in nocturnal surveys: Mirza zaza, Phaner parienti, Microcebussp. and Cheirogaleussp. with encounter rates of 1.2, 0.4, 0.4 and 0.3 individuals/km, respectively. The presence of Lepilemur dorsalis was confirmed by camera trap. This is the first time lemurs have been studied in cacao plantations, and understanding how these threatened animals use anthropogenic landscapes is vital for their conservation