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 polymerase chain reaction 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 axillary buds and the tuber-based bioassays. This suggests that S. endobioticum resistance genes are expressed in both etiolated "belowground" sprouts and green aboveground organs. RNAseq analysis demonstrated that the symptomatic aboveground materials contain less contaminants compared with 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. Because wart resistance is active in both below- and aboveground organs, the aboveground bioassay can potentially speed up screening for S. endobioticum resistance in potato breeding programs because 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. Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Generic RT-PCR tests for detection and identification of tospoviruses.

A set of tests for generic detection and identification of tospoviruses has been developed. Based on a multiple sequence alignment of the nucleocapsid gene and its 5' upstream untranslated region sequence from 28 different species, primers were designed for RT-PCR detection of tospoviruses from all recognized clades, i.e. the American, Asian and Eurasian clades, and from the small group of distinct and floating species. Pilot experiments on isolates from twenty different species showed that the designed primer sets successfully detected all species by RT-PCR, as confirmed by nucleotide sequence analysis of the amplicons. In a final optimized design, the primers were applied in a setting of five RT-PCR tests. Seven different tospoviruses were successfully identified from diagnostic samples and in addition a non-described tospovirus species from alstroemeria plants. The results demonstrate that the newly developed generic RT-PCR tests provide a relevant tool for broad detection and identification of tospoviruses in plant quarantine and diagnostic laboratories.

Real-time PCR Tests in Dutch Exotic Mosquito Surveys; Implementation of Aedes aegypti and Aedes albopictus Identification Tests, and the Development of Tests for the Identification of Aedes atropalpus and Aedes japonicus japonicus (Diptera: Culicidae).

Since 2009, The Netherlands Food and Consumer Product Safety Authority carries out surveys focusing on, amongst others, the presence of invasive mosquito species (IMS). Special attention is given to exotic container-breeding Aedes species Aedes aegypti (L.), Aedes albopictus (Skuse), Aedes atropalpus (Coquillett), and Aedes japonicus japonicus (Theobald). This study describes the implementation of real-time PCR tests described by Hill et al. (2008) for the identification of Ae. aegypti and Ae. albopictus, and the development of two novel real-time PCR tests for the identification of Ae. atropalpus and Ae. j. japonicus. Initial test showed that optimization of elements of the Ae. aegypti and Ae. albopictus tests was needed. Method validation tests were performed to determine if the implemented and newly developed tests are fit for routine diagnostics. Performance criteria of analytical sensitivity, analytical specificity, selectivity, repeatability, and reproducibility were determined. In addition, experiments were performed to determine the influence of environmental conditions on the usability of DNA extracted from mosquito specimens trapped in BG-Sentinel traps. The real-time PCR tests were demonstrated to be sensitive, specific, repeatable, reproducible, and are less prone to false negative results compared to partial cytochrome c oxidase I gene sequencing owing to the DNA fragmentation caused by environmental influences.

Development and validation of real-time PCR tests for the identification of four Spodoptera species: Spodoptera eridania, Spodoptera frugiperda, Spodoptera littoralis, and Spodoptera litura (Lepidoptera: Noctuidae).

The genus Spodoptera comprises 31 species, 4 of which are listed as quarantine pests for the European Union: Spodoptera eridania (Cramer), Spodoptera frugiperda (Smith), Spodoptera littoralis (Boisduval), and Spodoptera litura (F.). In international trade, the earlier life stages (eggs and larvae) are being intercepted at point of inspection most frequently, challenging the possibilities of morphological identification. To realize a rapid and reliable identification for all stages, we developed and validated four simplex real-time polymerase chain reaction identification tests based on the mitochondrial cytochrome b gene using dual-labeled hydrolysis probes. Method validation on dilutions of extracted DNA of the target organisms showed that low levels of template (up to 0.2-100 pg) can reliably be identified. No cross-reactivity was observed with 14 nontarget Spodoptera and 5 non-Spodoptera species in the specific Spodoptera tests. The tests showed to be repeatable, reproducible (both 100%), and robust. The new Spodoptera tests have proven to be suitable tools for routine identification of all life stages of S. eridania, S. frugiperda, S. littoralis, and S. litura.

Development and validation of a real-time RT-PCR assay for generic detection of pospiviroids.

In many countries phytosanitary regulations apply to Potato spindle tuber viroid, because it can cause serious diseases in potato and tomato crops. Other pospiviroids, some of which are distributed widely in ornamental crops, can cause similar diseases. Consequently, there is a need for a reliable and cost-effective generic testing method. An assay was developed that detects all known species of the genus Pospiviroid, using real-time RT-PCR based on TaqMan technology. This GenPospi assay consists of two reactions running in parallel, the first targeting all pospiviroids, except Columnea latent viroid, the second specifically targeting the latter viroid (already published). To monitor the RNA extraction a nad5 internal control was included. Method validation on tomato leaves showed that the GenPospi assay detects all pospiviroids up to a relative infection rate of 0.13% (equals 770 times dilution). The assay was specific because no cross reactivity was observed with other viroids, viruses or nucleic acid from plant hosts. Repeatability and reproducibility were 100% and the assay appeared robust in an inter-laboratory comparison. The GenPospi assay has been shown to be a suitable tool for large-scale screening for all known pospiviroids. Although it has been validated for tomato leaves it can potentially be used for any crop.