A powerful long metabarcoding method for the determination of complex diets from faecal analysis of the European pond turtle (Emys orbicularis, L. 1758).

High-throughput sequencing has become an accurate method for the identification of species present in soil, water, faeces, gut or stomach contents. However, information at the species level is limited due to the choice of short barcodes and based on the idea that DNA is too degraded to allow longer sequences to be amplified. We have therefore developed a long DNA metabarcoding method based on the sequencing of short reads followed by de novo assembly, which can precisely identify the taxonomic groups of organisms associated with complex diets, such as omnivorous individuals. The procedure includes 11 different primer pairs targeting the COI gene, the large subunit of the ribulose-1,5-bisphosphate carboxylase gene, the maturase K gene, the 28S rRNA and the trnL-trnF chloroplastic region. We validated this approach using 32 faeces samples from an omnivorous reptile, the European pond turtle (Emys orbicularis, L. 1758). This metabarcoding approach was assessed using controlled experiments including mock communities and faecal samples from captive feeding trials. The method allowed us to accurately identify prey DNA present in the diet of the European pond turtles to the species level in most of the cases (82.4%), based on the amplicon lengths of multiple markers (168-1,379 bp, average 546 bp), and produced by de novo assembly. The proposed approach can be adapted to analyse various diets, in numerous conservation and ecological applications. It is consequently appropriate for detecting fine dietary variations among individuals, populations and species as well as for the identification of rare food items

First report of Geosmithia langdonii and Geosmithia spp. isolated from a decaying elm (Ulmus minor) in Geneva, Switzerland

The mortality of a young elm Ulmus minor in 2014 in Geneva prompted a search for the microorganisms potentially involved. Symptoms included foliar chlorosis and wilting followed by defoliation of branches. Wood symptoms included a brown streaking of sap wood and brown stains in trunk and branches. The comparison of the resulting ITS rDNA sequences to the NCBI Nucleotide database allowed to identify 10 different organisms. The genus Geosmithia represented 48% of the isolates belonging to three species: Geosmithia langdonii (7 isolates) and 2 unknown morphologically and genetically different Geosmithia sp. 1 and sp. 2 (4 isolates). Geosmithia species are very little known ascomycetes, which have been recently shown to be opportunistic pathogens on broadleaved trees and conifers, living as saprobes in galleries of many bark beetle species. In the case described here, Geosmithia langdonii, and the unknown Geosmithia species were found in symptomatic wood while bark beetle galleries were found in close regions of the symptomatic wood. Geosmithia langdonii was the major fungus retrieved from the symptomatic wood and could have contributed, along with other identified fungal species, to a pathogenic complex producing symptoms similar to the ones of the Dutch Elm Disease and led to the dieback of this elm tree. Geosmithia langdonii and 2 yet unknown Geosmithia species (sp. 1 and sp. 2), different from any other reported Geosmithia species are reported from an elm tree in Switzerland for the first time

Biological control of the latent pathogen Gnomoniopsis smithogylvyi in European chestnut grafting scions using Bacillus amyloliquefaciens and Trichoderma atroviride

A search for endophytes in Castanea sativa Miller (Fagales: Fagaceae) grafting scions showed that a latent pathogenic fungus Gnomoniopsis smithogilvyi (Diaporthales: Gnomoniaceae) was present as the major component of the endophytic flora. Initially, the goal of this study was to develop a biological control method of Cryphonectria parasitica (Diaporthales: Valsaceae), the chestnut blight agent, by soaking chestnut scions before grafting in antagonists suspension. However, the healthy chestnut material used in in vitro and glasshouse experiments turned out to be naturally infected by a pathogen. At first view, the symptoms looked very similar to those caused by C. parasitica but some differences were noticed. DNA sequencing and application of Koch’s postulates revealed that G. smithogilvyi was the agent responsible of those symptoms. Preventive biocontrol experiments were carried out with chestnut tree scions soaked overnight in a liquid suspension of Bacillus amyloliquefaciens (Bacillales: Bacillaceae). This bacterium was then frequently found in the lower parts of scions (CF of 100% between 3.1 and 6 cm) and up to a height of 18 cm. It was observed that when B. amyloliquefaciens was present, the endophytic and opportunistic pathogenic fungus G. smithogilvyi was not present. Conversely, the parts not colonized by the bacteria were always naturally infected by the endophytic fungus. This would indicate that the endophytic behavior of B. amyloliquefaciens inhibited the growth of G. smithogilvyi and reduced its presence in scions. A similar experiment, carried out with the Trichoderma atroviride (Hypocreales: Hypocreaceae), led to similar observations. Trichoderma atroviride was frequently isolated in the lower parts of scions (CF of 100% until 6 cm) and up to a height of 27 cm. Inoculating B. amyloliquefaciens and T. atroviride as part of a preventive biocontrol treatment would allow these biological control agents to colonize the plant as endophytes and prevent the development of G. smithogilvyi