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

Live and dead qPCR detection demonstrates that feeding of Nosema ceranae results in infection in the honey bee but not the bumble bee

As the honey bee and bumble bee may suffer from the same or related microbial pathogens, cross contamination from commercially reared Bombus spp. to honey bees and wild bumble bees and vice versa is a major concern. Honey bee-collected pollen to feed commercially reared Bombus spp. is a potential risk. Nosema spp. is a fungal pathogen in bees. In this study, we developed new quantitative detection tools based on the detection of RNA using a TaqMan-based RT-qPCR for Nosema ceranae and Nosema apis, with extraction controls based on the actin gene of honey bees and bumble bees, respectively. These tools were subsequently applied to study the epidemiology of N. ceranae, a main disease in honey bees. We screened gamma radiation and cold treatment sterilisation for their efficacy to kill N. ceranae spores fed in sugar water and in pollen to honey bees and bumble bees, respectively. N. ceranae infection in adult bumble bees was checked. Spores passing the inter-alimentary track were found but no infection was observed. N. ceranae spores were fed to honey bees. Their presence and multiplication were demonstrated, showing the spores were both viable and infectious. Our results indicate that N. ceranae found in honey bees cannot infect commercially reared bumble bees (Bombus terrestris) and, that gamma radiation effectively kills N. ceranae. The highly specific and sensitive molecular assays developed, were exploited to detect N. ceranae in pollen and faeces, which would allow more comprehensive epidemiological studies on this important pathogen

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

The Synchytrium endobioticum AvrSen1 triggers a Hypersensitive Response in Sen1 potatoes while natural variants evade detection

Synchytrium endobioticum is an obligate biotrophic fungus of the phylum Chytridiomycota. It causes potato wart disease, has a world-wide quarantine status and is included on the HHS and USDA Select Agent list. S. endobioticum isolates are grouped in pathotypes based on their ability to evade host-resistance in a set of differential potato varieties. So far, thirty-nine pathotypes are reported. A single dominant gene (Sen1) governs pathotype 1 resistance and we anticipated that the underlying molecular model would involve a pathogen effector (AvrSen1) that is recognized by the host.The S. endobioticum specific secretome of fourteen isolates representing six different pathotypes was screened for effectors specifically present in pathotype 1(D1) isolates but absent in others. We discovered a single AvrSen1 candidate. Expression of this candidate in potato Sen1 plants showed a specific hypersensitive response, which co-segregated with the Sen1 resistance in potato populations. No HR was obtained with truncated genes found in pathotypes that evaded recognition by Sen1. These findings established that our candidate gene was indeed Avrsen1. AvrSen1 is a single copy gene and encodes a 376 amino acid protein without predicted function or functional domains, and is the first effector gene identified in Chytridiomycota

The linear mitochondrial genome of the quarantine chytrid Synchytrium endobioticum; insights into the evolution and recent history of an obligate biotrophic plant pathogen

Species of the Chytridiomycota, also known as chytrids, belong to a basal lineage in the fungal kingdom inhabiting terrestrial and aquatic environments. Most of the described chytrids are free-living saprophytes, but several species cause important diseases. Examples are Batrachochytrium dendrobatidis responsible for the world wide amphibian decline and Synchytrium endobioticum the causal agent of potato wart disease. Synchytrium endobioticum has an obligate biotrophic lifestyle and isolates can be differentiated based on their virulence on a differential set of potato cultivars, referred to as pathotypes. Quarantine measures have been implemented worldwide to control the disease and to prevent its spread. To determine taxonomical relationships, and to gain insights into the evolution and recent history of introductions of this plant pathogen we assembled and annotated the complete mitochondrial genome of S. endobioticum and generated mitochondrial genomes for five additional chytrid species. The mitochondrial genome of S. endobioticum pathotype 1(D1) strain MB42 is a linear 72,865 bp molecule with terminal inverted repeats that encodes 14 core genes typically found on fungal mitochondrial genomes. Based on single nucleotide polymorphisms the 30 S. endobioticum isolates sequenced could be clustered in four distinct mitochondrial lineages, indicating multiple introductions of the pest to the European main land. These lineages comprise different pathotypes suggesting that pathotypes 2(G1) and 6(O1) have emerged at least twice independently. Variations for polymorphic sites within a strain were observed demonstrating that S. endobioticum strains represent in fact a community of different genotypes. Such a community was shown to be complex and stable over time, but we also demonstrate that the population may shift rapidly based on selection for virulence on a specific R gene from the host

The linear mitochondrial genome of the quarantine chytrid Synchytrium endobioticum; insights into the evolution and recent history of an obligate biotrophic plant pathogen

Species of the Chytridiomycota, also known as chytrids, belong to a basal lineage in the fungal kingdom inhabiting terrestrial and aquatic environments. Most of the described chytrids are free-living saprophytes, but several species cause important diseases. Examples are Batrachochytrium dendrobatidis responsible for the world wide amphibian decline and Synchytrium endobioticum the causal agent of potato wart disease. Synchytrium endobioticum has an obligate biotrophic lifestyle and isolates can be differentiated based on their virulence on a differential set of potato cultivars, referred to as pathotypes. Quarantine measures have been implemented worldwide to control the disease and to prevent its spread. To determine taxonomical relationships, and to gain insights into the evolution and recent history of introductions of this plant pathogen we assembled and annotated the complete mitochondrial genome of S. endobioticum and generated mitochondrial genomes for five additional chytrid species. The mitochondrial genome of S. endobioticum pathotype 1(D1) strain MB42 is a linear 72,865 bp molecule with terminal inverted repeats that encodes 14 core genes typically found on fungal mitochondrial genomes. Based on single nucleotide polymorphisms the 30 S. endobioticum isolates sequenced could be clustered in four distinct mitochondrial lineages, indicating multiple introductions of the pest to the European main land. These lineages comprise different pathotypes suggesting that pathotypes 2(G1) and 6(O1) have emerged at least twice independently. Variations for polymorphic sites within a strain were observed demonstrating that S. endobioticum strains represent in fact a community of different genotypes. Such a community was shown to be complex and stable over time, but we also demonstrate that the population may shift rapidly based on selection for virulence on a specific R gene from the host

The Synchytrium endobioticum AvrSen1 triggers a Hypersensitive Response in Sen1 potatoes while natural variants evade detection

Synchytrium endobioticum is an obligate biotrophic fungus of the phylum Chytridiomycota. It causes potato wart disease, has a world-wide quarantine status and is included on the HHS and USDA Select Agent list. S. endobioticum isolates are grouped in pathotypes based on their ability to evade host-resistance in a set of differential potato varieties. So far, thirty-nine pathotypes are reported. A single dominant gene (Sen1) governs pathotype 1 resistance and we anticipated that the underlying molecular model would involve a pathogen effector (AvrSen1) that is recognized by the host.The S. endobioticum specific secretome of fourteen isolates representing six different pathotypes was screened for effectors specifically present in pathotype 1(D1) isolates but absent in others. We discovered a single AvrSen1 candidate. Expression of this candidate in potato Sen1 plants showed a specific hypersensitive response, which co-segregated with the Sen1 resistance in potato populations. No HR was obtained with truncated genes found in pathotypes that evaded recognition by Sen1. These findings established that our candidate gene was indeed Avrsen1. AvrSen1 is a single copy gene and encodes a 376 amino acid protein without predicted function or functional domains, and is the first effector gene identified in Chytridiomycota

The linear mitochondrial genome of the quarantine chytrid Synchytrium endobioticum; insights into the evolution and recent history of an obligate biotrophic plant pathogen

Species of the Chytridiomycota, also known as chytrids, belong to a basal lineage in the fungal kingdom inhabiting terrestrial and aquatic environments. Most of the described chytrids are free-living saprophytes, but several species cause important diseases. Examples are Batrachochytrium dendrobatidis responsible for the world wide amphibian decline and Synchytrium endobioticum the causal agent of potato wart disease. Synchytrium endobioticum has an obligate biotrophic lifestyle and isolates can be differentiated based on their virulence on a differential set of potato cultivars, referred to as pathotypes. Quarantine measures have been implemented worldwide to control the disease and to prevent its spread. To determine taxonomical relationships, and to gain insights into the evolution and recent history of introductions of this plant pathogen we assembled and annotated the complete mitochondrial genome of S. endobioticum and generated mitochondrial genomes for five additional chytrid species. The mitochondrial genome of S. endobioticum pathotype 1(D1) strain MB42 is a linear 72,865 bp molecule with terminal inverted repeats that encodes 14 core genes typically found on fungal mitochondrial genomes. Based on single nucleotide polymorphisms the 30 S. endobioticum isolates sequenced could be clustered in four distinct mitochondrial lineages, indicating multiple introductions of the pest to the European main land. These lineages comprise different pathotypes suggesting that pathotypes 2(G1) and 6(O1) have emerged at least twice independently. Variations for polymorphic sites within a strain were observed demonstrating that S. endobioticum strains represent in fact a community of different genotypes. Such a community was shown to be complex and stable over time, but we also demonstrate that the population may shift rapidly based on selection for virulence on a specific R gene from the host

The linear mitochondrial genome of the quarantine chytrid Synchytrium endobioticum; insights into the evolution and recent history of an obligate biotrophic plant pathogen

Chytridiomycota species (chytrids) belong to a basal lineage in the fungal kingdom. Inhabiting terrestrial and aquatic environments, most are free-living saprophytes but several species cause important diseases: e.g. Batrachochytrium dendrobatidis, responsible for worldwide amphibian decline; and Synchytrium endobioticum, causing potato wart disease. S. endobioticum has an obligate biotrophic lifestyle and isolates can be further characterized as pathotypes based on their virulence on a differential set of potato cultivars. Quarantine measures have been implemented globally to control the disease and prevent its spread. We used a comparative approach using chytrid mitogenomes to determine taxonomical relationships and to gain insights into the evolution and recent history of introductions of this plant pathogen. Results We assembled and annotated the complete mitochondrial genome of 30 S. endobioticum isolates and generated mitochondrial genomes for five additional chytrid species. The mitochondrial genome of S. endobioticum is linear with terminal inverted repeats which was validated by tailing and PCR amplifying the telomeric ends. Surprisingly, no conservation in organisation and orientation of mitochondrial genes was observed among the Chytridiomycota except for S. endobioticum and its sister species Synchytrium microbalum. However, the mitochondrial genome of S. microbalum is circular and comprises only a third of the 72.9 Kbp found for S. endobioticum suggesting recent linearization and expansion. Four mitochondrial lineages were identified in the S. endobioticum mitochondrial genomes. Several pathotypes occur in different lineages, suggesting that these have emerged independently. In addition, variations for polymorphic sites in the mitochondrial genome of individual isolates were observed demonstrating that S. endobioticum isolates represent a community of different genotypes. Such communities were shown to be complex and stable over time, but we also demonstrate that the use of semi-resistant potato cultivars triggers a rapid shift in the mitochondrial haplotype associated with increased virulence. Conclusions Mitochondrial genomic variation shows that S. endobioticum has been introduced into Europe multiple times, that several pathotypes emerged multiple times, and that isolates represent communities of different genotypes. Our study represents the most comprehensive dataset of chytrid mitogenomes, which provides new insights into the extraordinary dynamics and evolution of mitochondrial genomes involving linearization, expansion and reshuffling

Large scale identification of rodenticide resistance in Rattus norvegicus and Mus musculus in the Netherlands based on Vkorc1 codon 139 mutations.

Background: Resistance to rodenticides has been reported globally and poses a considerable problem for efficacy of pest control. The most documented resistance to rodenticides in commensal rodents is associated with mutations in the Vkorc1 gene,in particular in codon 139. Resistance to anticoagulant rodenticides has been reported in the Netherlands since 1989. A study from 2013 showed that 25% of 169 Norway rats (Rattus norvegicus) had a mutation at codon 139 of the Vkorc1 gene. To gain insight in the current status of rodenticide resistance amongst R. norvegicus and M. musculus in the Netherlands , we tested Norway rats and house mice (Mus musculus) for mutations in codon 139 of the Vkorc1 gene. In addition, we collected data from pest controllers on their use of rodenticides and experience with rodenticide resistance.Results: A total of 1,801 rodent samples were collected throughout the country consisting of 1404 R. norvegicus and 397 M. musculus. In total, 15% of R. norvegicus (95% CI: 13 - 17%) and 38% of M. musculus (95% CI: 33 - 43%) carried a genetic mutation at codon 139 of the Vkorc1 gene.Conclusion: This study demonstrates genetic mutations at codon 139 of the Vkorc1 gene in M. musculus in the Netherlands. Resistance to anticoagulant rodenticides is present in R. norvegicus and M. musculus in multiple regions in the Netherlands. The results of this comprehensive study provide a baseline and facilitate trend analyses of Vkorc1 codon 139 mutations and evaluation of integrated pest management (IPM) strategies as these are enrolled in the Netherlands