On the species status of the root-knot nematode Meloidogyne ulmi Palmisano & Ambrogioni, 2000 (Nematoda, Meloidogynidae)

The root-knot nematode Meloidogyne ulmi is synonymised with Meloidogyne mali based on morphological and morphometric similarities, common hosts, as well as biochemical similarities at both protein and DNA levels. M. mali was first described in Japan on Malus prunifolia Borkh.; and M. ulmi in Italy on Ulmus chenmoui W.C. Cheng. Morphological and morphometric studies of their holo- and paratypes revealed important similarities in the major characters as well as some general variability in a few others. Host test also showed that besides the two species being able to parasitize the type hosts of the other, they share some other common hosts. Our study of the esterase and malate dehydrogenase isozyme phenotypes of some M. ulmi populations gave a perfectly comparable result to that already known for M. mali. Finally, phylogenetic studies of their SSU and LSU rDNA sequence data revealed that the two are not distinguishable at DNA level. All these put together, leave strong evidences to support the fact that M. ulmi is not a valid species, but a junior synonym of M. mali. Brief discussion on the biology and life cycle of M. mali is given. An overview of all known hosts and the possible distribution of M. mali in Europe are also presented

First Expansion of the Public Tomato Brown Rugose Fruit Virus (ToBRFV) Nextstrain Build; Inclusion of New Genomic and Epidemiological Data

Tomato brown rugose fruit virus (ToBRFV) is a tobamovirus that was first detected in Israel and Jordan following an outbreak of a new disease infecting tomato in 2014. Since then, the virus has been reported from all continents except Oceania and Antarctica. In response to the first finding of the virus in The Netherlands, the Dutch National Plant Protection Organization created a ToBRFV Nextstrain build (v1). In this report, we announce 47 new (near) complete ToBRFV genomes and the generation of the new ToBRFV Nextstrain (v2) build containing 118 ToBRFV genomes with associated geographic and epidemiological data. Examples of utilization of the genomic sequences are presented, and we report the first sequence from South America and present a novel hypothesis on the possible ToBRFV center of origin.[Graphic: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license

An alternative bioassay for Synchytrium endobioticum demonstrates the expression of potato wart resistance in aboveground plant parts

The obligate biotrophic chytrid species Synchytrium endobioticum is the causal agent of potato wart disease. Currently 39 pathotypes have been described based on their interaction with a differential set of potato varieties. Wart resistance and pathotyping is performed using bioassays in which etiolated tuber sprouts are inoculated. Here we describe an alternative method in which aboveground plant parts are inoculated. Susceptible plants produced typical wart symptoms in developing, but not in fully expanded, aboveground organs. Colonization of the host by S endobioticum was verified by screening for resting spores by microscopy and by molecular techniques using TaqMan PCR and RNAseq analysis. When applied to resistant plants, none of these symptoms were detectable. Recognition of S. endobioticum pathotypes by differentially resistant potato varieties was identical in aboveground plant parts and the tuber-based bioassays. This suggests that S. endobioticum resistance genes are expressed both in etiolated “belowground” sprouts and green aboveground organs. RNAseq analysis demonstrated that the symptomatic aboveground materials contain less contaminants compared to resting spores extracted from tuber-based assays. This reduced microbial contamination in the aboveground bioassay could be an important advantage to study this obligate biotrophic plant-pathogen interaction. As wart resistance is active in both below and above ground organs, the aboveground bioassay can potentially speed up screening for S. endobioticum resistance in potato breeding programs as it omits the requirement for tuber formation. In addition, possibilities arise to express S. endobioticum effectors in potato leaves through agroinfiltration, thereby providing additional phenotyping tools for research and breeding

An integrated approach for synonymization of Rotylenchus rhomboides with R. goodeyi (Nematoda: Hoplolaimidae) reveals high intraspecific mitogenomic variation

Rotylenchus is a widely-distributed economically important plant-parasitic nematode group whose species-level identification relies largely on limited morphological characters including character-based tabular keys and molecular data of ribosomal and mitochondrial genes. In this study, a combined morphological and molecular analysis of three populations of R. goodeyi from Belgium, Poland and the Netherlands revealed important character variations of this species leading to synonymisation of R. rhomboides with R. goodeyi, and a high nucleotide variation within cox1 gene sequences in these populations. Additional Illumina sequencing of DNA from individuals of the Dutch population revealed two variants of mitogenomes each of approximately 23 Kb in size, differing by about 9% and containing eleven protein coding genes, two ribosomal RNA genes and up to 29 transfer RNA genes. In addition to the first representative whole genome shotgun sequence datasets of the genus Rotylenchus, this study also provides the full length mitogenome and the ribosomal DNA sequences of R. goodeyi

An alternative bioassay for Synchytrium endobioticum demonstrates the expression of potato wart resistance in aboveground plant parts

The obligate biotrophic chytrid species Synchytrium endobioticum is the causal agent of potato wart disease. Currently 39 pathotypes have been described based on their interaction with a differential set of potato varieties. Wart resistance and pathotyping is performed using bioassays in which etiolated tuber sprouts are inoculated. Here we describe an alternative method in which aboveground plant parts are inoculated. Susceptible plants produced typical wart symptoms in developing, but not in fully expanded, aboveground organs. Colonization of the host by S endobioticum was verified by screening for resting spores by microscopy and by molecular techniques using TaqMan PCR and RNAseq analysis. When applied to resistant plants, none of these symptoms were detectable. Recognition of S. endobioticum pathotypes by differentially resistant potato varieties was identical in aboveground plant parts and the tuber-based bioassays. This suggests that S. endobioticum resistance genes are expressed both in etiolated “belowground” sprouts and green aboveground organs. RNAseq analysis demonstrated that the symptomatic aboveground materials contain less contaminants compared to resting spores extracted from tuber-based assays. This reduced microbial contamination in the aboveground bioassay could be an important advantage to study this obligate biotrophic plant-pathogen interaction. As wart resistance is active in both below and above ground organs, the aboveground bioassay can potentially speed up screening for S. endobioticum resistance in potato breeding programs as it omits the requirement for tuber formation. In addition, possibilities arise to express S. endobioticum effectors in potato leaves through agroinfiltration, thereby providing additional phenotyping tools for research and breeding

Real-time tracking of Tomato brown rugose fruit virus (ToBRFV) outbreaks in the Netherlands using Nextstrain.

Tomato brown rugose fruit virus (ToBRFV) is a Tobamovirus that was first observed in 2014 and 2015 on tomato plants in Israel and Jordan respectively. Since the first description, the virus has been reported from all continents except Oceania and Antarctica, and has been found infecting both tomato and pepper crops. In October 2019, the Dutch National Plant Protection Organization received a ToBRFV infected tomato sample as part of a generic survey targeting tomato pests. Presence of the virus was verified using Illumina sequencing. A follow-up survey was initiated to determine the extent of ToBRFV presence in the Dutch tomato horticulture and identify possible linkages between ToBRFV genotypes, companies and epidemiological traits. Nextstrain was used to visualize these potential connections. By November 2019, 68 companies had been visited of which 17 companies were found to be infected. The 50 ToBRFV genomes from these outbreak locations group in three main clusters, which are hypothesized to represent three original sources. No correlation was found between genotypes, companies and epidemiological traits, and the source(s) of the Dutch ToBRFV outbreak remain unknown. This paper describes a Nextstrain build containing ToBRFV genomes up to and including November 2019. Sharing data with this interactive online tool will enable the plant virology field to better understand and communicate the diversity and spread of this new virus. Organizations are invited to share data or materials for inclusion in the Nextstrain build, which can be accessed at https://nextstrain.nrcnvwa.nl/ToBRFV/20191231

Analysis of Thaumatotibia leucotreta (Lepidoptera: Tortricidae: Olethreutinae) mitochondrial genomes in the context of a recent host range expansion

Abstract Background The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick, 1913), is a significant pest of various important economic crops and is a EU quarantine pest. In the last decade the pest has been reported on Rosa spp. In this study we determined whether this shift occurred within specific FCM populations across seven eastern sub-Saharan countries or whether the species opportunistically switches to this novel host as it presents itself. To achieve this, we assessed the genetic diversity of complete mitogenomes of T. leucotreta specimens intercepted at import and analysed potential linkages with the geographical origin and host species. Results Genomic, geographical and host information were integrated into a T. leucotreta Nextstrain build which contains 95 complete mitogenomes generated from material intercepted at import between January 2013 and December 2018. Samples represented seven sub-Saharan countries and mitogenomic sequences grouped in six main clades. Discussion If host strains of FCM would exist, specialization from a single haplotype towards the novel host is expected. Instead, we find specimens intercepted on Rosa spp. in all six clades. The absence of linkage between genotype and host suggests opportunistic expansion to the new host plant. This underlines risks of introducing new plant species to an area as the effect of pests already present on the new plant might be unpredictable with current knowledge

Tracking outbreak populations of the pepper weevil Anthonomus eugenii (Coleoptera; Curculionidae) using complete mitochondrial genomes

The pepper weevil, Anthonomus eugenii, is a major pest on Capsicum species. Apart from natural spread, there is a risk of spread via international pepper trade. In the Netherlands, a pepper weevil outbreak occurred in 2012 and affected six greenhouses producing different sweet pepper varieties. The following year, a pepper weevil outbreak occurred in Italy. To trace the origin of the Dutch outbreak and to establish if the Dutch and Italian outbreaks were linked, we determined the mitogenomes of A. eugenii specimens collected at outbreak locations, and compared these with specimens from the native area, and other areas where the pest was introduced either by natural dispersal or via trade. The circular 17,257 bp A. eugenii mitogenome comprises thirteen mitochondrial genes typically found in insect species. Intra-species variation of these mitochondrial genes revealed four main mitochondrial lineages encompassing 41 haplotypes. The highest diversity was observed for specimens from its presumed native area (i.e. Mexico). The Dutch outbreak specimens represented three highly similar haplotypes, suggesting a single introduction of the pest. The major Dutch haplotype was also found in two specimens from New Jersey. As the Netherlands does not have pepper trade with New Jersey, it is likely that the specimens sampled in New Jersey and those sampled in the Netherlands originate from a shared source that was not included in this study. In addition, our analysis shows that the Italian and Dutch outbreaks were not linked. The mitochondrial genome is a useful tool to trace outbreak populations and the methodology presented in this paper could prove valuable for other invasive pest species, such as the African fruit moth Thaumatotibia leucotreta and emerald ash borer Agrilus planipennis.</p

Imported Hyalomma ticks in the Netherlands 2018-2020.

Background: Ticks of the genus Hyalomma, which are vectors for several tick-borne diseases, are occasionally found in areas outside their endemic range including northern parts of Europe. The objective of this study was to analyse adult Hyalomma ticks that were recently found in the Netherlands. Methods: Hyalomma ticks were morphologically identified. Cluster analysis, based upon sequence data (cox1 barcoding) for molecular identification, and pathogen detection were performed. Additionally, a cross-sectional survey of horses was conducted to actively search for Hyalomma ticks in summer 2019. Analysis of temperature was done to assess the possibility of (i) introduced engorged nymphs moulting to adults and (ii) establishment of populations in the Netherlands. Results: Seventeen adult Hyalomma ticks (one in 2018, eleven in 2019, five in 2020) were found by citizens and reported. Fifteen ticks were detected on horses and two on humans. Twelve were identified as H. marginatum, one as H. rufipes and four, of which only photographic images were available, as Hyalomma sp. No Crimean-Congo haemorrhagic fever virus or Babesia/Theileria parasites were detected. One adult tick tested positive for Rickettsia aeschlimannii. In the cross-sectional horse survey, no Hyalomma ticks were found. Analysis of temperatures showed that engorged nymphs arriving on migratory birds in spring were able to moult to adults in 2019 and 2020, and that cumulative daily temperatures in the Netherlands were lower than in areas with established H. marginatum populations. Conclusions: Our results show that Hyalomma ticks are regularly introduced in the Netherlands as nymphs. Under the Dutch weather conditions, these nymphs are able to develop to the adult stage, which can be sighted by vigilant citizens. Only one human pathogen, Rickettsia aeschlimannii, was found in one of the ticks. The risk of introduction of tick-borne diseases via Hyalomma ticks on migratory birds is considered to be low. Establishment of permanent Hyalomma populations is considered unlikely under the current Dutch climatic conditions.[Figure not available: see fulltext.