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 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

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

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

Development and Validation of a High-Throughput Sequencing Test for Mitogenome and rDNA Assembly and Annotation, and Its Use in Support of Nematode Identification of Regulatory Concern

Nematoda is a diverse phylum, and representatives are found in most habitats, including in and on animals and plants. Nematodes are regarded as the most abundant group in terms of individuals in marine and terrestrial sediments. Plant-parasitic nematodes are globally responsible for an annual yield loss of $125 billion. Reliable species identification is essential to take appropriate phytosanitary measures. The introduction of validated Sanger sequencing of 18S, 28S, and cox1 barcode loci represented a powerful tool in support of nematode identification. However, technical challenges associated with PCR and Sanger sequencing and the need for additional loci for identification hamper the efficient use of sequence data. To overcome these challenges, we developed an automated bioinformatic pipeline for the assembly and annotation of mitochondrial genomes and ribosomal DNAs, and we defined and validated a standardized test protocol including controls for routine diagnostics (i.e., high-throughput sequencing [HTS] test). The HTS test can be performed on single nematode specimens and outperforms the Sanger-based sequencing by producing less ambiguous consensus sequences and by yielding additional sequence data offering additional diagnostic resolution when needed. Compared with Sanger sequencing, the HTS test represents a reduction in hands-on time. The HTS test is regarded as fit for the purpose of the molecular identification of single nematode specimens in support of nematode diagnostics of regulatory concern. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license