Development and evaluation of one-step RT-qPCR TaqMan multiplex panels applied to six viruses occurring in lily and tulip bulbs.

One-step RT-qPCR TaqMan assays have been developed for six plant viruses with considerable economic impact in the growing of tulip and lily bulbs: lily mottle virus, lily symptomless virus, lily virus X, Plantago asiatica mosaic virus, tulip breaking virus and tulip virus X. To enhance efficacy and cost-efficiency these assays were combined into multiplex panels. Four different multiplex panels were designed, each consisting of three virus assays and an adapted assay for the housekeeping gene nad5 of lilies and tulips, that acts as an internal amplification control. To eliminate false negative results due to variation in the viral genome sequences, for each target virus two assays were developed on distinct conserved genomic regions. Specificity, PCR efficiency and compatibility of primers and probes were tested using gBlock constructions. Diagnostic samples were used to evaluate the strategy. High Throughput Sequencing of a set of the diagnostic samples, further verified the presence or absence of the viruses in the RNA samples and sequence variations in the target genes. This interchangeable multiplex panel strategy may be a valuable tool for the detection of viruses in certification, surveys and virus diagnostics.

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 .

Population dynamics of Phytophthora infestans in the Netherlands reveals expansion and spread of dominant clonal lineages and virulence in sexual offspring.

For a comprehensive survey of the structure and dynamics of the Dutch Phytophthora infestans population, 652 P. infestans isolates were collected from commercial potato fields in the Netherlands during the 10-year period 2000-2009. Genotyping was performed using 12 highly informative microsatellite markers and mitochondrial haplotypes. In addition, for each isolate, the mating type was determined. STRUCTURE analysis grouped the 322 identified genotypes in three clusters. Cluster 1 consists of a single clonal lineage NL-001, known as "Blue_13"; all isolates in this cluster have the A2 mating type and the Ia mitochondrial haplotype. Clusters 2 and 3 display a more elaborate substructure containing many unique genotypes. In Cluster 3, several distinct clonal lineages were also identified. This survey witnesses that the Dutch population underwent dramatic changes in the 10 years under study. The most notable change was the emergence and spread of A2 mating type strain NL-001 (or "Blue_13"). The results emphasize the importance of the sexual cycle in generating genetic diversity and the importance of the asexual cycle as the propagation and dispersal mechanism for successful genotypes. Isolates were also screened for absence of the Avrblb1/ipiO class I gene, which is indicative for virulence on Rpi-blb1. This is also the first report of Rpi-blb1 breakers in the Netherlands. Superimposing the virulence screening on the SSR genetic backbone indicates that lack the Avrblb1/ipiO class I gene only occurred in sexual progeny. So far, the asexual spread of the virulent isolates identified has been limited.

Accurate quantification of microorganisms in PCR-inhibiting environmental DNA extracts by a novel internal amplification control approach using Biotrove OpenArrays.

PCR-based detection assays are prone to inhibition by substances present in environmental samples, thereby potentially leading to inaccurate target quantification or false-negative results. Internal amplification controls (IACs) have been developed to help alleviate this problem but are generally applied in a single concentration, thereby yielding less-than-optimal results across the wide range of microbial gene target concentrations possible in environmental samples (J. Hoorfar, B. Malorny, A. Abdulmawjood, N. Cook, M. Wagner, and P. Fach, J. Clin. Microbiol. 42:1863-1868, 2004). Increasing the number of IACs for each quantitative PCR (qPCR) sample individually, however, typically reduces sensitivity and, more importantly, the reliability of quantification. Fortunately, current advances in high-throughput qPCR platforms offer the possibility of multiple reactions for a single sample simultaneously, thereby allowing the implementation of more than one IAC concentration per sample. Here, we describe the development of a novel IAC approach that is specifically designed for the state-of-the-art Biotrove OpenArray platform. Different IAC targets were applied at a range of concentrations, yielding a calibration IAC curve for each individual DNA sample. The developed IACs were optimized, tested, and validated by using more than 5,000 unique qPCR amplifications, allowing accurate quantification of microorganisms when applied to soil DNA extracts containing various levels of PCR-inhibiting compounds. To our knowledge, this is the first study using a suite of IACs at different target concentrations to monitor PCR inhibition across a wide target range, thereby allowing reliable and accurate quantification of microorganisms in PCR-inhibiting DNA extracts. The developed IAC is ideally suited for high-throughput screenings of, for example, ecological and agricultural samples on next-generation qPCR platforms.

Physiological and molecular responses of Lactuca sativa to colonization by Salmonella enterica serovar Dublin.

This paper describes the physiological and molecular interactions between the human-pathogenic organism Salmonella enterica serovar Dublin and the commercially available mini Roman lettuce cv. Tamburo. The association of S. enterica serovar Dublin with lettuce plants was first determined, which indicated the presence of significant populations outside and inside the plants. The latter was evidenced from significant residual concentrations after highly efficient surface disinfection (99.81%) and fluorescence microscopy of S. enterica serovar Dublin in cross sections of lettuce at the root-shoot transition region. The plant biomass was reduced significantly compared to that of noncolonized plants upon colonization with S. enterica serovar Dublin. In addition to the physiological response, transcriptome analysis by cDNA amplified fragment length polymorphism analysis also provided clear differential gene expression profiles between noncolonized and colonized lettuce plants. From these, generally and differentially expressed genes were selected and identified by sequence analysis, followed by reverse transcription-PCR displaying the specific gene expression profiles in time. Functional grouping of the expressed genes indicated a correlation between colonization of the plants and an increase in expressed pathogenicity-related genes. This study indicates that lettuce plants respond to the presence of S. enterica serovar Dublin at physiological and molecular levels, as shown by the reduction in growth and the concurrent expression of pathogenicity-related genes. In addition, it was confirmed that Salmonella spp. can colonize the interior of lettuce plants, thus potentially imposing a human health risk when processed and consumed.

Secretory granule proteins from the subventral esophageal glands of the potato cyst nematode identified by monoclonal antibodies to a protein fraction from second-stage juveniles.

Sodium dodecyl sulfate-extracted proteins from second-stage juveniles (J2) of the potato cyst nematode Globodera rostochiensis were fractionated by preparative continuous flow electrophoresis, and monoclonal antibodies (MAbs) were raised against the 38- to 40.5-kDa protein fraction. Screening of the hybridoma culture fluids by immunofluorescence microscopy of J2 resulted in the identification of 12 MAbs that bound specifically to the subventral esophageal glands. On Western blots of J2 these MAbs identified four protein bands with apparent molecular masses of 30, 31, 39, and 49 kDa. Immunoelectron microscopy with one of these MAbs showed an intense labeling of the electron dense core of the secretory granules in the subventral gland cells of J2. It is concluded that one or more of these proteins are localized within these secretory granules. Immunofluorescence microscopy of J2 from other plant parasitic nematode species showed that most of these MAbs also bind to the subventral glands of G. pallida and G. tabacum but not of Heterodera schachtii, H. glycines, Meloidogyne incognita, or M. hapla.