Matthew Dickinson

Rapid Detection of Phytophthora ramorum and P. kernoviae by Two-Minute DNA Extraction Followed by Isothermal Amplification and Amplicon Detection by Generic Lateral Flow Device

ABSTRACT A method for nucleic-acid-based detection of pathogens in plant material has been developed which comprises a simple and rapid method for extracting DNA on the nitrocellulose membranes of lateral-flow devices, loop-mediated isothermal amplification (LAMP) of target DNA using labeled primers, and detection of the generically labeled amplification products by a sandwich immunoassay in a lateral-flow-device format. Each of these steps can be performed without specialist equipment and is suitable for on-site use, and a result can be obtained in just over an hour. A LAMP assay for the detection of plant DNA (cytochrome oxidase gene) can be used in conjunction with pathogen-specific assays to confirm negative results. The use of this method is demonstrated for the detection of Phytophthora ramorum, the causal agent of sudden oak death and dieback/leaf blight in a range of tree, shrub, and herbaceous species, and the recently described pathogen P. kernoviae.

Interaction of Escherichia coli with growing salad spinach plants.

In this study, the interaction of a bioluminescence-labeled Escherichia coli strain with growing spinach plants was assessed. Through bioluminescence profiles, the direct visualization of E. coli growing around the roots of developing seedlings was accomplished. Subsequent in situ glucuronidase (GUS) staining of seedlings confirmed that E. coli had become internalized within root tissue and, to a limited extent, within hypocotyls. When inoculated seeds were sown in soil microcosms and cultivated for 42 days, E. coli was recovered from the external surfaces of spinach roots and leaves as well as from surface-sterilized roots. When 20-day-old spinach seedlings (from uninoculated seeds) were transferred to soil inoculated with E. coli, the bacterium became established on the plant surface, but internalization into the inner root tissue was restricted. However, for seedlings transferred to a hydroponic system containing 10(2) or 10(3) CFU of E. coli per ml of the circulating nutrient solution, the bacterium was recovered from surface-sterilized roots, indicating that it had been internalized. Differences between E. coli interactions in the soil and those in the hydroponic system may be attributed to greater accessibility of the roots in the latter model. Alternatively, the presence of a competitive microflora in soil may have restricted root colonization by E. coli. The implications of this studys findings with regard to the microbiological safety of minimally processed vegetables are discussed.

Detection of Botrytis cinerea by loop-mediated isothermal amplification.

Aims:  To develop a sensitive, rapid and simple method for detection of Botrytis cinerea based on loop‐mediated isothermal amplification (LAMP) that would be suitable for use outside a conventional laboratory setting.

Genetic differentiation of the Aspergillus section Flavi complex using AFLP fingerprints.

Twenty-four isolates of Aspergillus sojae, A. parasiticus, A. oryzae and A. flavus, including a number that have the capacity to produce aflatoxin, have been compared using amplified fragment length polymorphisms (AFLPs). Based on analysis of 12 different primer combinations, 500 potentially polymorphic fragments have been identified. Analysis of the AFLP data consistently and clearly separates the A. sojae/A. parasiticus isolates from the A. oryzae/A. flavus isolates. Furthermore. there are markers that can be used to distinguish the A. sojae isolates from those of A. parasiticus, which form the basis for species-specific markers. However, whilst there were many polymorphisms between isolates within the A. oryzae/A. flavus subgroup, no markers could be identified that distinguish between the two species. Sequencing of the ribosomal DNA ITS (internal transcribed spacers) from selected isolates also separated the A. sojae/A. parasiticus subgroup from the A. oryzae/A. flavus subgroup, but was unable to distinguish between the A. sojae and A. parasiticus isolates. Some ITS variation was found between isolates within the A. oryzae/A. flavus subgroup, but did not correlate with the species classification, indicating that it is difficult to use molecular data to separate the two species. In addition, sequencing of ribosomal ITS regions and AFLP analysis suggested that some species annotations in public culture collections may be inaccurate.

Isolation of genes expressed during compatible interactions between leaf rust (Puccinia triticina) and wheat using cDNA-AFLP.

SUMMARY The rust fungi are obligate biotrophic pathogens that depend on living host tissue for their growth. In compatible interactions they go through a number of developmental stages to form intercellular hyphae and haustoria within host cells, through which they obtain their nutrients. Here we have exploited the cDNA-AFLP technique to isolate fragments of wheat and rust genes that are expressed at specific defined time-points during the infection process. A number of these sequences were used as probes in Northern hybridizations and in RT-PCR to confirm their expression patterns, and were also characterized by PCR analysis and Southern hybridizations to determine whether they are of fungal or wheat origin. A cDNA library was constructed from pooled RNAs extracted from days 5 and 7 after inoculation, and this library was screened to isolate full-length cDNAs of selected sequences. Sequence analysis of these cDNA fragments and clones revealed similarities amongst the fungal genes to a chitinase, a sorbitol utilization protein, an arabinitol dehydrogenase and a proteasome regulatory unit, whilst in wheat, we identified sequences with homology to a katanin and a cell enlargement protein.

Internalization of Human Pathogens within Growing Salad Vegetables

’Department of Food Science, University of Guelph, Guelph, Ontario, Canada NIG 2W1, 2Division of Food Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughbm.ough, Leicestershire, LEI2 5RD, UK, ’Division of Plant Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LEI2 5RD, UK and 4Division of Agricultural Sciences, School of Biosciences. University of Nottingham, Sutton Bonington Canzpus, Loughborough, Leicestershire, LEI2 5RD, UK

Panel of 23S rRNA Gene-Based Real-Time PCR Assays for Improved Universal and Group-Specific Detection of Phytoplasmas†

ABSTRACT Primers and probes based on the 23S rRNA gene have been utilized to design a range of real-time PCR assays for routine phytoplasma diagnostics. These assays have been authenticated as phytoplasma specific and shown to be at least as sensitive as nested PCR. A universal assay to detect all phytoplasmas has been developed, along with a multiplex assay to discriminate 16SrI group phytoplasmas from members of all of the other 16Sr groups. Assays for the 16SrII, 16SrIV, and 16SrXII groups have also been developed to confirm that the 23S rRNA gene can be used to design group-specific assays.

LAMP assay and rapid sample preparation method for on‐site detection of flavescence dorée phytoplasma in grapevine

In Europe the most devastating phytoplasma associated with grapevine yellows (GY) diseases is a quarantine pest, flavescence dorée (FDp), from the 16SrV taxonomic group. The on-site detection of FDp with an affordable device would contribute to faster and more efficient decisions on the control measures for FDp. Therefore, a real-time isothermal LAMP assay for detection of FDp was validated according to the EPPO standards and MIQE guidelines. The LAMP assay was shown to be specific and extremely sensitive, because it detected FDp in all leaf samples that were determined to be FDp infected using quantitative real-time PCR. The whole procedure of sample preparation and testing was designed and optimized for on-site detection and can be completed in one hour. The homogenization procedure of the grapevine samples (leaf vein, flower or berry) was optimized to allow direct testing of crude homogenates with the LAMP assay, without the need for DNA extraction, and was shown to be extremely sensitive.

Gene discovery in EST sequences from the wheat leaf rust fungus Puccinia triticina sexual spores, asexual spores and haustoria, compared to other rust and corn smut fungi

BackgroundRust fungi are biotrophic basidiomycete plant pathogens that cause major diseases on plants and trees world-wide, affecting agriculture and forestry. Their biotrophic nature precludes many established molecular genetic manipulations and lines of research. The generation of genomic resources for these microbes is leading to novel insights into biology such as interactions with the hosts and guiding directions for breakthrough research in plant pathology.ResultsTo support gene discovery and gene model verification in the genome of the wheat leaf rust fungus, Puccinia triticina (Pt), we have generated Expressed Sequence Tags (ESTs) by sampling several life cycle stages. We focused on several spore stages and isolated haustorial structures from infected wheat, generating 17,684 ESTs. We produced sequences from both the sexual (pycniospores, aeciospores and teliospores) and asexual (germinated urediniospores) stages of the life cycle. From pycniospores and aeciospores, produced by infecting the alternate host, meadow rue (Thalictrum speciosissimum), 4,869 and 1,292 reads were generated, respectively. We generated 3,703 ESTs from teliospores produced on the senescent primary wheat host. Finally, we generated 6,817 reads from haustoria isolated from infected wheat as well as 1,003 sequences from germinated urediniospores. Along with 25,558 previously generated ESTs, we compiled a database of 13,328 non-redundant sequences (4,506 singlets and 8,822 contigs). Fungal genes were predicted using the EST version of the self-training GeneMarkS algorithm. To refine the EST database, we compared EST sequences by BLASTN to a set of 454 pyrosequencing-generated contigs and Sanger BAC-end sequences derived both from the Pt genome, and to ESTs and genome reads from wheat. A collection of 6,308 fungal genes was identified and compared to sequences of the cereal rusts, Puccinia graminis f. sp. tritici (Pgt) and stripe rust, P. striiformis f. sp. tritici (Pst), and poplar leaf rust Melampsora species, and the corn smut fungus, Ustilago maydis (Um). While extensive homologies were found, many genes appeared novel and species-specific; over 40% of genes did not match any known sequence in existing databases. Focusing on spore stages, direct comparison to Um identified potential functional homologs, possibly allowing heterologous functional analysis in that model fungus. Many potentially secreted protein genes were identified by similarity searches against genes and proteins of Pgt and Melampsora spp., revealing apparent orthologs.ConclusionsThe current set of Pt unigenes contributes to gene discovery in this major cereal pathogen and will be invaluable for gene model verification in the genome sequence.

Loop-mediated isothermal amplification for rapid detection of the causal agents of cassava brown streak disease

The causal agents of cassava brown streak disease have recently been identified as Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Primers have been developed for rapid detection of these viruses by reverse transcription loop-mediated isothermal amplification (RT-LAMP). Performance of the RT-LAMP assays compared favourably with published RT-PCR and real-time RT-PCR methods. Furthermore, amplification by RT-LAMP is completed in 40 min and does not require thermal cycling equipment. Modification of the RT-LAMP reactions to use labelled primers allowed rapid detection of amplification products using lateral flow devices containing antibodies specific to the incorporated labels, avoiding the need for fluorescence detection or gel electrophoresis.