A Panel of Real-Time PCR Assays for the Direct Detection of All of the Xylella fastidiosa Subspecies.

The bacterial plant pathogen Xylella fastidiosa causes disease in hundreds of plant species worldwide including many crops, and as such accurate determination of the subspecies of the bacteria is vital to control, containment, and eradication measures. Conventional methods to determine the subspecies of X. fastidiosa rely on time consuming multilocus sequence typing (MLST), a laborious multistage process. This chapter provides a rapid alternative to MLST utilizing real-time PCR assays to provide highly specific and sensitive detection of the pathogen subspecies. Here we describe the methodology for sampling plant material, performing the DNA extraction and undertaking the real-time PCR assays. This method allows straightforward, robust, reliable, high-throughput, and rapid determination of the X. fastidiosa subspecies.

Six Multiplex TaqManTM-qPCR Assays for Quantitative Diagnostics of Pseudomonas Species Causative of Bacterial Blotch Diseases of Mushrooms.

Bacterial blotch is a group of economically important diseases of the common button mushroom (Agaricus bisporus). Once the pathogens are introduced to a farm, mesophilic growing conditions (that are optimum for mushroom production) result in severe and widespread secondary infections. Efficient, timely and quantitative detection of the pathogens is hence critical for the design of localized control strategies and prediction of disease risk. This study describes the development of real-time TaqManTM assays that allow molecular diagnosis of three currently prevalent bacterial blotch pathogens: "Pseudomonas gingeri," Pseudomonas tolaasii and (as yet uncharacterized) Pseudomonas strains (belonging to Pseudomonas salomonii and Pseudomonas edaphica). For each pathogen, assays targeting specific DNA markers on two different loci, were developed for primary detection and secondary verification. All six developed assays showed high diagnostic specificity and sensitivity when tested against a panel of 63 Pseudomonas strains and 40 other plant pathogenic bacteria. The assays demonstrated good analytical performance indicated by linearity across calibration curve (>0.95), amplification efficiency (>90%) and magnitude of amplification signal (>2.1). The limits of detection were optimized for efficient quantification in bacterial cultures, symptomatic tissue, infected casing soil and water samples from mushroom farms. Each target assay was multiplexed with two additional assays. Xanthomonas campestris was detected as an extraction control, to account for loss of DNA during sample processing. And the total Pseudomonas population was detected, to quantify the proportion of pathogenic to beneficial Pseudomonas in the soil. This ratio is speculated to be an indicator for blotch outbreaks. The multiplexed assays were successfully validated and applied by routine testing of diseased mushrooms, peat sources, casing soils, and water from commercial production units.

Rapid Sample Preparation and LAMP for Phytoplasma Detection.

Loop-mediated isothermal AMPlification (LAMP) allows the rapid detection of pathogens by polymerase-mediated amplification of target nucleic acid sequences at a single incubation temperature. LAMP can be combined with very simple sample preparation/crude DNA extraction protocols, allowing the method to be used away from the laboratory for in-field detection. Equally, these benefits can also be leveraged to provide a rapid method suited to high-throughput diagnostic laboratories. In this chapter we described a crude DNA extraction protocol suitable for use in the field and provide a protocol for real-time detection using LAMP.

Development of Loop-Mediated Isothermal Amplification Assays for the Detection of Seedborne Fungal Pathogens Fusarium fujikuroi and Magnaporthe oryzae in Rice Seed.

Bakanae disease (caused by Fusarium fujikuroi) and rice blast (caused by Magnaporthe oryzae) are two of the most important seedborne pathogens of rice. The detection of both pathogens in rice seed is necessary to maintain high quality standards and avoid production losses. Currently, blotter tests are used followed by morphological identification of the developing pathogens to provide an incidence of infection in seed lots. Two loop-mediated isothermal amplification assays were developed with primers designed to target the elongation factor 1-α sequence of F. fujikuroi and the calmodulin sequence of M. oryzae. The specificity, sensitivity, selectivity, repeatability, and reproducibility for each assay was assessed in line with the international validation standard published by the European and Mediterranean Plant Protection Organization (PM7/98). The results showed a limit of detection of 100 to 999 fg of DNA of F. fujikuroi and 10 to 99 pg of M. oryzae DNA. When combined with a commercial DNA extraction kit, the assays were demonstrated to be effective for use in detection of the pathogens in commercial batches of infected rice seed of different cultivars, giving results equivalent to the blotter method, thus demonstrating the reliability of the method for the surveillance of F. fujikuroi and M. oryzae in seed-testing laboratories.

The invasion, provenance and diversity of Vespa velutina Lepeletier (Hymenoptera: Vespidae) in Great Britain.

The yellow-legged or Asian hornet (Vespa velutina colour form nigrithorax) was introduced into France from China over a decade ago. Vespa velutina has since spread rapidly across Europe, facilitated by suitable climatic conditions and the ability of a single nest to disperse many mated queens over a large area. Yellow-legged hornets are a major concern because of the potential impact they have on populations of many beneficial pollinators, most notably the western honey bee (Apis mellifera), which shows no effective defensive behaviours against this exotic predator. Here, we present the first report of this species in Great Britain. Actively foraging hornets were detected at two locations, the first around a single nest in Gloucestershire, and the second a single hornet trapped 54 km away in Somerset. The foraging activity observed in Gloucestershire was largely restricted to within 700 m of a single nest, suggesting highly localised movements. Genetic analyses of individuals from the Gloucestershire nest and the single hornet from Somerset suggest that these incursions represent an expansion of the European population, rather than a second incursion from Asia. The founding queen of the Gloucestershire nest mated with a single male, suggesting that sexual reproduction may have occurred in an area of low nest density. Whilst the nest contained diploid adult males, haploid 'true' males were only present at the egg stage, indicating that the nest was detected and removed before the production of queens. Members of the public reported additional dead hornets associated with camping equipment recently returned from France and imported timber products, highlighting possible pathways of incursion. The utility of microsatellites to inform surveillance during an incursion and the challenge of achieving eradication of this damaging pest are discussed.

DNA barcoding for biosecurity: case studies from the UK plant protection program.

Since its conception, DNA barcoding has seen a rapid uptake within the research community. Nevertheless, as with many new scientific tools, progression towards the point of routine deployment within diagnostic laboratories has been slow. In this paper, we discuss the application of DNA barcoding in the Defra plant health diagnostic laboratories, where DNA barcoding is used primarily for the identification of invertebrate pests. We present a series of case studies that demonstrate the successful application of DNA barcoding but also reveal some potential limitations to expanded use. The regulated plant pest, Bursephalenchus xylophilus, and one of its vectors, Monochamus alternatus, were found in dining chairs. Some traded wood products are potentially high risk, allowing the movement of longhorn beetles; Trichoferus campestris, Leptura quadrifasciata, and Trichoferus holosericeus were found in a wooden cutlery tray, a railway sleeper, and a dining chair, respectively. An outbreak of Meloidogyne fallax was identified in Allium ampeloprasum and in three weed species. Reference sequences for UK native psyllids were generated to enable the development of rapid diagnostics to be used for monitoring following the release of Aphalara itadori as a biological control agent for Fallopia japonica.

Discovery of an unknown diversity of Leucinodes species damaging Solanaceae fruits in sub-Saharan Africa and moving in trade (Insecta, Lepidoptera, Pyraloidea).

The larvae of the Old World genera Leucinodes Guenée, 1854 and Sceliodes Guenée, 1854 are internal feeders in the fruits of Solanaceae, causing economic damage to cultivated plants like Solanummelongena and Solanumaethiopicum. In sub-Saharan Africa five nominal species of Leucinodes and one of Sceliodes occur. One of these species, the eggplant fruit and shoot borer Leucinodesorbonalis Guenée, 1854, is regarded as regularly intercepted from Africa and Asia in Europe, North and South America and is therefore a quarantine pest on these continents. We investigate the taxonomy of African Leucinodes and Sceliodes based on morphological characters in wing pattern, genitalia and larvae, as well as mitochondrial DNA, providing these data for identification of all life stages. The results suggest that both genera are congeneric, with Sceliodes syn. n. established as junior subjective synonym of Leucinodes. Leucinodesorbonalis is described from Asia and none of the samples investigated from Africa belong to this species. Instead, sub-Saharan Africa harbours a complex of eight endemic Leucinodes species. Among the former nominal species of Leucinodes (and Sceliodes) from Africa, only Leucinodeslaisalis (Walker, 1859), comb. n. (Sceliodes) is confirmed, with Leucinodestranslucidalis Gaede, 1917, syn. n. as a junior subjective synonym. The other African Leucinodes species were unknown to science and are described as new: Leucinodesafricensis sp. n., Leucinodesethiopica sp. n., Leucinodeskenyensis sp. n., Leucinodesmalawiensis sp. n., Leucinodespseudorbonalis sp. n., Leucinodesrimavallis sp. n. and Leucinodesugandensis sp. n. An identification key based on male genitalia is provided for the African Leucinodes species. Most imports of Leucinodes specimens from Africa into Europe refer to Leucinodesafricensis, which has been frequently imported with fruits during the last 50 years. In contrast, Leucinodeslaisalis has been much less frequently recorded, and Leucinodespseudorbonalis as well as Leucinodesrimavallis only very recently in fruit imports from Uganda. Accordingly, interceptions of Leucinodes from Africa into other continents will need to be re-investigated for their species identity and will likely require, at least in parts, revisions of the quarantine regulations. The following African taxa are excluded from Leucinodes: Hyperanalyta Strand, 1918, syn. rev. as revised synonym of Analyta Lederer, 1863; Analytaapicalis (Hampson, 1896), comb. n. (Leucinodes); Lygropiaaureomarginalis (Gaede, 1916), comb. n. (Leucinodes); Sylleptehemichionalis Mabille, 1900, comb. rev., Sylleptehemichionalisidalis Viette, 1958, comb. rev. and Sylleptevagans (Tutt, 1890), comb. n. (Aphytoceros). Deanolisiriocapna (Meyrick, 1938), comb. n. from Indonesia is originally described and misplaced in Sceliodes, and Leucinodescordalis (Doubleday, 1843), comb. n. (Margaritia) from New Zealand, Leucinodesraondry (Viette, 1981), comb. n. (Daraba) from Madagascar as well as Leucinodesgrisealis (Kenrick, 1912), comb. n. (Sceliodes) from New Guinea are transferred from Sceliodes to Leucinodes. While Leucinodes is now revised from Africa, it still needs further revision in Asia.

The development of monoclonal antibodies to the secA protein of Cape St. Paul wilt disease phytoplasma and their evaluation as a diagnostic tool.

Partial recombinant secA proteins were produced from six different phytoplasma isolates representing five 16Sr groups and the expressed, purified recombinant (partial secA) protein from Cape St. Paul wilt disease phytoplasma (CSPWD, 16SrXXII) was used to immunise mice. Monoclonal antibodies (mAbs) were selected by screening hybridoma supernatants for binding to the recombinant proteins. To characterise the binding to proteins from different phytoplasmas, the antibodies were screened by ELISA and western blotting, and epitope mapping was undertaken. Eight different mAbs with varying degrees of specificity against recombinant proteins from different phytoplasma groups were selected. Western blotting revealed that the mAbs bind to proteins in infected plant material, two of which were specific for phytoplasmas. ELISA testing of infected material, however, gave negative results suggesting that either secA was not expressed at sufficiently high levels, or conformational changes of the reagents adversely affected detection. This work has shown that the phytoplasma secA gene is not a suitable antibody target for routine detection, but has illustrated proof of principle for the methodology.

The phytoplasmas: an introduction.

This volume of "Methods in Molecular Biology" entitled "Phytoplasmas: Methods and Protocols" aims to provide a broad range of protocols for working with this group of plant pathogens. In this first chapter, we provide some background information about the phytoplasmas to put the protocols into context.

Techniques for the maintenance and propagation of phytoplasmas in glasshouse collections of Catharanthus roseus.

Phytoplasma collections are a vital resource for researchers and diagnosticians studying phytoplasma diseases. They provide material as a point of reference and a research tool to increase our understanding of phytoplasmas and the diseases they cause. This chapter describes the techniques required to create and maintain collections of phytoplasma-infected Catharanthus roseus (Madagascar periwinkle).

PCR analysis of phytoplasmas based on the secA gene.

Conventionally, diagnostics and phylogenetics of phytoplasmas have been primarily based on the 16S rRNA gene, for which "universal" primers are available that amplify from most phytoplasma 16Sr groups. However, there has been a drive in recent years to develop "universal" primers for other genes that can be used to complement the use of the 16S rRNA gene. This chapter details the use of primers based on the phytoplasma secA gene and describes how these primers can be used in both a single or nested PCR approach for amplification. It also notes the use of appropriate controls that should be undertaken and provides a source for phytoplasma secA sequences that are available in databases that can be used for phylogenetic analyses.

T-RFLP for detection and identification of phytoplasmas in plants.

Conventionally, detection of phytoplasmas has been performed by PCR of the 16S rRNA gene, followed by either RFLP or DNA sequencing to determine the phytoplasma 16Sr group. This chapter demonstrates the technique of terminal restriction fragment length polymorphism (T-RFLP), a fingerprinting technique which combines both detection and identification in a single method, with the added benefit of inbuilt controls which removes the risk of false negative results and in addition highlights potential false positive results.

Panel of 23S rRNA gene-based real-time PCR assays for improved universal and group-specific detection of phytoplasmas.

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.

Phytoplasma phylogenetics based on analysis of secA and 23S rRNA gene sequences for improved resolution of candidate species of 'Candidatus Phytoplasma'.

Phytoplasma phylogenetics has focused primarily on sequences of the non-coding 16S rRNA gene and the 16S-23S rRNA intergenic spacer region (16-23S ISR), and primers that enable amplification of these regions from all phytoplasmas by PCR are well established. In this study, primers based on the secA gene have been developed into a semi-nested PCR assay that results in a sequence of the expected size (about 480 bp) from all 34 phytoplasmas examined, including strains representative of 12 16Sr groups. Phylogenetic analysis of secA gene sequences showed similar clustering of phytoplasmas when compared with clusters resolved by similar sequence analyses of a 16-23S ISR-23S rRNA gene contig or of the 16S rRNA gene alone. The main differences between trees were in the branch lengths, which were elongated in the 16-23S ISR-23S rRNA gene tree when compared with the 16S rRNA gene tree and elongated still further in the secA gene tree, despite this being a shorter sequence. The improved resolution in the secA gene-derived phylogenetic tree resulted in the 16SrII group splitting into two distinct clusters, while phytoplasmas associated with coconut lethal yellowing-type diseases split into three distinct groups, thereby supporting past proposals that they represent different candidate species within 'Candidatus Phytoplasma'. The ability to differentiate 16Sr groups and subgroups by virtual RFLP analysis of secA gene sequences suggests that this gene may provide an informative alternative molecular marker for pathogen identification and diagnosis of phytoplasma diseases.