Population genetic diversity and dynamics of the honey bee brood pathogen Melissococcus plutonius in a region with high prevalence.

European foulbrood (EFB) is a honey bee brood disease caused by the bacterium Melissococcus plutonius. Large-scale EFB outbreaks have been reported in several countries in recent decades, which entail costly sanitation measures of affected apiaries to restrict the spread of this contagious pathogen. To mitigate its impact, a better understanding of the population dynamics of the etiological agent is required. We here used multi-locus sequence typing (MLST) to infer the genetic diversity and geographical distribution of 160 M. plutonius isolates collected from EFB symptomatic honey bee colonies seven years apart. Isolates belonged to three clonal complexes (CCs) known worldwide and to 12 sequence types (STs), of which five were novel. Phylogenetic and clustering analyses showed that some of these novel sequence types have likely evolved locally during a period of outbreak, but most disappeared again. We further screened the isolates for melissotoxin A (mtxA), a putative virulence gene. The prevalence of STs in which mtxA was frequent increased over time, suggesting that this gene promotes spread. Despite the increased frequency of this gene in the population, the total number of cases decreased, which could be due to stricter control measures implemented before the second sampling period. Our results provide a better understanding of M. plutonius population dynamics and help identify knowledge gaps that limit efficient control of this emerging disease.

Molecular characterization of Pseudomonas from Agaricus bisporus caps reveal novel blotch pathogens in Western Europe.

BACKGROUND: Bacterial blotch is a group of economically important diseases affecting the cultivation of common button mushroom, Agaricus bisporus. Despite being studied for more than a century, the identity and nomenclature of blotch-causing Pseudomonas species is still unclear. This study aims to molecularly characterize the phylogenetic and phenotypic diversity of blotch pathogens in Western Europe. METHODS: In this study, blotched mushrooms were sampled from farms across the Netherlands, United Kingdom and Belgium. Bacteria were isolated from symptomatic cap tissue and tested in pathogenicity assays on fresh caps and in pots. Whole genome sequences of pathogenic and non-pathogenic isolates were used to establish phylogeny via multi-locus sequence alignment (MLSA), average nucleotide identity (ANI) and in-silico DNA:DNA hybridization (DDH) analyses. RESULTS: The known pathogens "Pseudomonas gingeri", P. tolaasii, "P. reactans" and P. costantinii were recovered from blotched mushroom caps. Seven novel pathogens were also identified, namely, P. yamanorum, P. edaphica, P. salomonii and strains that clustered with Pseudomonas sp. NC02 in one genomic species, and three non-pseudomonads, i.e. Serratia liquefaciens, S. proteamaculans and a Pantoea sp. Insights on the pathogenicity and symptom severity of these blotch pathogens were also generated. CONCLUSION: A detailed overview of genetic and regional diversity and the virulence of blotch pathogens in Western Europe, was obtained via the phylogenetic and phenotypic analyses. This information has implications in the study of symptomatic disease expression, development of diagnostic tools and design of localized strategies for disease management.

Assessment of Surface Sterilisation Approaches for the Removal of Pollen DNA from Philaenus spumarius.

Dietary analysis of herbivorous insects relies on successfully eliminating surface contamination. If this cannot be performed reliably, then it will not be possible to differentiate between plants that the insect is feeding on and plants the insect has been in contact with, either directly or via pollen. Methods in the literature often use bleach and alcohol washes to remove contamination. We perform a controlled metabarcoding baseline study on a herbivorous, xylem-feeding insect, the Meadow Spittlebug (Philaenus spumarius), using Oxford Nanopore Technologies (ONT) sequencing, and identify possible contamination that persists after washes. Despite the reported success of methods in the literature, we find that contamination is still present, leading to possible false-positive results. We hypothesise that pollen is the main source of contamination, its robust nature making it difficult to remove, and conduct a further three experiments with the goal of removing pollen from the surface of Philaenus spumarius. This study investigates the effectiveness of robust bleach/Tween/alcohol washes, sterile gut excision (including combined with Distel application), and ultraviolet light as alternative sterilisation approaches. Overall, our findings indicate that we are unable to remove surface contamination and still detect signals that may originate in the gut. In no experiment did we unequivocally detect plant DNA that originated in the P. spumarius gut.

Development and validation of a multi-locus DNA metabarcoding method to identify endangered species in complex samples.

DNA metabarcoding provides great potential for species identification in complex samples such as food supplements and traditional medicines. Such a method would aid Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. A DNA metabarcoding method was developed that makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa and that facilitate the identification of species in samples containing degraded DNA. The DNA metabarcoding method was developed based on Illumina MiSeq amplicon sequencing of well-defined experimental mixtures, for which a bioinformatics pipeline with user-friendly web-interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. The advanced multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES-listed species. The method can provide improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to an enhanced quality assurance.

Molecular epidemiology and population structure of the honey bee brood pathogen Melissococcus plutonius.

Melissococcus plutonius is the causative agent of European foulbrood (EFB), which is a serious brood disease of the European honey bee (Apis mellifera). EFB remains a threat because of a poor understanding of disease epidemiology. We used a recently published multi-locus sequence typing method to characterise 206 M. plutonius isolates recovered from outbreaks in England and Wales over the course of 2 years. We detected 15 different sequence types (STs), which were resolved by eBURST and phylogenetic analysis into three clonal complexes (CCs) 3, 12 and 13. Single and double locus variants within CC3 were the most abundant and widespread genotypes, accounting for 85% of the cases. In contrast, CCs 12 and 13 were rarer and predominantly found in geographical regions of high sampling intensity, consistent with a more recent introduction and localised spread. K-function analysis and interpoint distance tests revealed significant geographical clustering in five common STs, but pointed to different dispersal patterns between STs. We noted that CCs appeared to vary in pathogenicity and that infection caused by the more pathogenic variants is more likely to lead to honey bee colony destruction, as opposed to treatment. The importance of these findings for improving our understanding of disease aetiology and control are discussed.

A typing scheme for the honeybee pathogen Melissococcus plutonius allows detection of disease transmission events and a study of the distribution of variants.

Melissococcus plutonius is the bacterial pathogen that causes European Foulbrood of honeybees, a globally important honeybee brood disease. We have used next-generation sequencing to identify highly polymorphic regions in an otherwise genetically homogenous organism, and used these loci to create a modified MLST scheme. This synthesis of a proven typing scheme format with next-generation sequencing combines reliability and low costs with insights only available from high-throughput sequencing technologies. Using this scheme we show that the global distribution of M.plutonius variants is not uniform. We use the scheme in epidemiological studies to trace movements of infective material around England, insights that would have been impossible to confirm without the typing scheme. We also demonstrate the persistence of local variants over time.