Comparative genomics of chytrid fungi reveal insights into the obligate biotrophic and pathogenic lifestyle of Synchytrium endobioticum.

Synchytrium endobioticum is an obligate biotrophic soilborne Chytridiomycota (chytrid) species that causes potato wart disease, and represents the most basal lineage among the fungal plant pathogens. We have chosen a functional genomics approach exploiting knowledge acquired from other fungal taxa and compared this to several saprobic and pathogenic chytrid species. Observations linked to obligate biotrophy, genome plasticity and pathogenicity are reported. Essential purine pathway genes were found uniquely absent in S. endobioticum, suggesting that it relies on scavenging guanine from its host for survival. The small gene-dense and intron-rich chytrid genomes were not protected for genome duplications by repeat-induced point mutation. Both pathogenic chytrids Batrachochytrium dendrobatidis and S. endobioticum contained the largest amounts of repeats, and we identified S. endobioticum specific candidate effectors that are associated with repeat-rich regions. These candidate effectors share a highly conserved motif, and show isolate specific duplications. A reduced set of cell wall degrading enzymes, and LysM protein expansions were found in S. endobioticum, which may prevent triggering plant defense responses. Our study underlines the high diversity in chytrids compared to the well-studied Ascomycota and Basidiomycota, reflects characteristic biological differences between the phyla, and shows commonalities in genomic features among pathogenic fungi.

Development of Polymorphic Microsatellite Loci for Potato Wart from Next-Generation Sequence Data.

Synchytrium endobioticum is the fungal agent causing potato wart disease. Because of its severity and persistence, quarantine measures are enforced worldwide to avoid the spread of this disease. Molecular markers exist for species-specific detection of this pathogen, yet markers to study the intraspecific genetic diversity of S. endobioticum were not available. Whole-genome sequence data from Dutch pathotype 1 isolate MB42 of S. endobioticum were mined for perfect microsatellite motifs. Of the 62 selected microsatellites, 21 could be amplified successfully and displayed moderate levels of polymorphism in 22 S. endobioticum isolates from different countries. Nineteen multilocus genotypes were observed, with only three isolates from Canada displaying identical profiles. The majority of isolates from Canada clustered genetically. In contrast, most isolates collected in Europe show no genetic clustering associated with their geographic origin. S. endobioticum isolates with the same pathotype displayed highly variable genotypes and none of the microsatellite markers correlated with a specific pathotype. The markers developed in this study can be used to assess intraspecific genetic diversity of S. endobioticum and allow track and trace of genotypes that will generate a better understanding of the migration and spread of this important fungal pathogen and support management of this disease.

Phylogeny of the genus Synchytrium and the development of TaqMan PCR assay for sensitive detection of Synchytrium endobioticum in soil.

Potato wart, caused by the fungal pathogen Synchytrium endobioticum, is a serious disease with the potential to cause significant economic damage. The small subunit (SSU) and internal transcribed spacer (ITS) ribosomal DNA (rDNA) were sequenced for several Synchytrium spp., showing a high rate of variability for both of these markers among the different species and monophyly of the genus within phylum Chytridiomycota. The intergenic nontranscribed spacer (IGS) of rDNA was sequenced for different pathotypes and showed no intraspecific variation within S. endobioticum, similar to the other rDNA markers from this study. To facilitate screening for the pathogen in soil, three TaqMan polymerase chain reaction (PCR) assays were developed from SSU, ITS, and IGS rDNA sequences to detect S. endobioticum sporangia in the chloroform-flotation fraction of sieved soil extracts. In the screening portion of the method, a first TaqMan assay targeting the SSU rDNA was developed with positive results that were further confirmed with amplicon melt analysis. A synthetic reaction control cloned into a plasmid was incorporated into the procedure, facilitating the validation of negative results. The presence of the reaction control did not adversely affect the efficiency of the SSU target amplification. A second TaqMan assay targeting the ITS-1 region was developed as a confirmatory test. There was 100% accordance between the SSU and ITS-1 TaqMan assays. Utilizing these two assays in tandem achieved good specificity for S. endobioticum, generating negative results with the cloned SSU and ITS-1 regions from all 14 other Synchytrium spp. considered. Spike recovery experiments indicated that these assays, targeting the SSU and ITS-1 rDNA regions, developed from a phylogeny dataset of the genus, could reliably detect a single sporangium in the chloroform flotation fraction of a soil extract. Good correlation between microscopic detection of sporangia and PCR results in both positive and negative soil samples was dually demonstrated for both the SSU and ITS-1 assays.

An Internal Reaction Control for Routine Detection of Clavibacter michiganensis subsp. sepedonicus Using a Real-Time TaqMan PCR-Based Assay.

An internal reaction control was integrated into a TaqMan polymerase chain reaction (PCR) assay for the detection of Clavibacter michiganensis subsp. sepedonicus, the causal organism of bacterial ring rot of potato. The reaction control, cloned into plasmid pCmsC4, consisted of a sequence unrelated to C. michiganensis subsp. sepedonicus flanked by the primer sequences used in the TaqMan PCR, thus eliminating the need for multiplexing. Inclusion of the reaction control plasmid in the TaqMan assay had no effect on either the limit of detection or the specificity of the method. Addition of SYBR Green permitted melt analysis of PCR products. The 242-bp reaction control amplicon, with a melt temperature of approximately 94.5°C, could easily be distinguished from the 152-bp primary diagnostic target amplicon, which had a melt temperature of about 85.5°C. Electrophoretic analysis showed that appearance of either melt peak correlated well with the presence of the appropriate amplicon. Two different substances, guanidine-HCl and humic acid, inhibited the amplification of the reaction control at concentrations lower than those that inhibited the primary diagnostic target, demonstrating the reaction control's effectiveness in detecting inhibition or reaction failure. Using the reaction control plasmid, a quantitative threshold for inhibitor detection was established. This permitted the validation of negative results, and thus facilitated the use of TaqMan real-time PCR in the routine testing of diagnostic samples for C. michiganensis subsp. sepedonicus.

Comparison of several methods for the extraction of DNA from potatoes and potato-derived products.

Eight methods were compared for the extraction of DNA from raw potato tubers, and nine methods were evaluated for the extraction of DNA from dehydrated potato slices, potato flakes, potato flour, potato starch, and two ready-to-eat potato snack foods. Extracts were assessed for yield using a fluorescence-based DNA quantification assay. Real-time amplification of an endogenous gene, sucrose synthase (sus), was used to assess extract and template quality. A CTAB-based method extracted the highest DNA yields from the tuber material. An in-house method, which utilized the Kingfisher magnetic particle processor, yielded the highest template quality from the tubers. For most of the tuber samples, the Kingfisher and CTAB methods recovered the highest levels of amplifiable sus. DNA yields for potato-derived foods generally decreased with the extent that the product had been processed. The methods that utilized the magnetic particle processor delivered the highest template quality from one of the snack products that was particularly high in fat. For most of the remaining processed products, the levels of amplifiable target DNA recovered were roughly correlated with total DNA recovery, indicating that overall yield had greater influence over sus amplification than template quality. The Wizard method was generally the best method for the extraction of DNA from most of the potato-derived foods.

Method for the detection of synthetic cry3A in transgenic potatoes.

All transgenic cultivars of potatoes registered in Canada and the United States have been modified to express a synthetic cry3A gene as a means of conferring resistance against the Colorado potato beetle, an important economic pest of potatoes. A PCR method was developed to amplify a 499 bp region of the synthetic cry3A gene. Using this method, synthetic cry3A could be detected in six different transgenic cultivars. Positive results could be confirmed with PvuII restriction digestion of the PCR-generated amplicon, which resulted in two fragments that were 283 and 216 bp in size. Of the 52 tuber extracts tested with this method, no false positive or false negative results were obtained, suggesting the method could be used with a high degree of accuracy. The absolute limit of detection was the number of cry3A copies present in one or perhaps two haploid copies of the potato genome. The practical limit of detection in tubers on a fresh weight basis was 0.02% for the NL 10-SUP and 0.01% for the remaining cultivars. Synthetic cry3A could also be detected in processed food products such as potato chips, shoestring potatoes, and frozen French fries. The method was suitable for screening potato tuber lots and some processed foods for the presence of synthetic cry3A.