Genomics informed design of a suite of real-time PCR assays for the specific detection of each Xylella fastidiosa subspecies.

AIMS: Existing methods for the identification of the subspecies of Xylella fastidiosa are time-consuming which can lead to delays in diagnosis and the associated plant health response to outbreaks and interceptions. METHODS AND RESULTS: Diagnostic markers were identified using a comparative genomics approach to allow fine differentiation of the very closely related subspecies. Five qPCR assays were designed to allow specific detection of X. fastidiosa subsp. fastidiosa, X. fastidiosa subsp. multiplex, X. fastidiosa subsp. pauca, X. fastidiosa subsp. morus and X. fastidiosa subsp. sandyi. All assays were validated according to the European and Mediterranean Plant Protection Organisation (EPPO) standard PM7/98(2). CONCLUSIONS: All of the assays were shown to be specific to the target subspecies and all the assays could be used to detect femtogram quantities of X. fastidiosa DNA. SIGNIFICANCE AND IMPACT OF THE STUDY: At present, diagnosing the subspecies of X. fastidiosa requires multiple conventional PCR assays (although only available for three of the five subspecies) or multi-locus sequence typing which takes several days. By comparison, the new assays provide a substantial reduction in the turnaround time for direct identification to the subspecies level in as little as 75 min.

Rapid, specific, simple, in-field detection of Xanthomonas campestris pathovar musacearum by loop-mediated isothermal amplification.

AIMS: To develop and evaluate a loop-mediated isothermal amplification (LAMP) assay for Xanthomonas campestris pathovar musacearum (Xcm), the causal agent of banana Xanthomonas wilt, a major disease of banana in Africa. METHODS AND RESULTS: LAMP primers were designed to the general secretion pathway protein D gene and tested against 17 isolates of Xcm encompassing the known genetic and geographic diversity of the bacterium and all isolates were detected. Seventeen other Xanthomonas isolates, including closely related Xanthomonas vasicola, other bacterial pathogens/endophytes of Musa and two healthy Musa varieties gave negative results with the LAMP assay. The assay showed good sensitivity, detecting as little as 51 fg of Xcm DNA, a greater level of sensitivity than that of an Xcm PCR assay. Amplification with the LAMP assay was very rapid, typically within 9 min from bacterial cultures. Symptomatic field samples of Musa from Uganda were tested and all produced amplification in less than 13 min. CONCLUSIONS: The LAMP assay provides rapid, sensitive detection of the pathogen that is ideally suited for deployment in laboratories with basic facilities and in-field situations. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first LAMP assay for Xcm which provides a significant improvement compared to existing diagnostics.

The complete genome sequence of Piper yellow mottle virus (PYMoV).

This study reports the first complete genome sequence of Piper yellow mottle virus (PYMoV, KC808712) identified in black pepper. The genome is 7,622 nucleotides long, possessing four open reading frames (ORFs). ORF1, ORF2 and ORF4 of PYMoV are reported as hypothetical proteins of unknown function with a predicted molecular mass of 15.7, 17.1 and 17.9 kDa, respectively. ORF3 of PYMoV encodes a polyprotein of 218.6 kDa and consists of a viral movement protein (MP), trimeric dUTPase, zinc finger, retropepsin, RT-LTR, and RNAse H. Detailed PYMoV genome analysis confirmed that it is a member of the family Caulimoviridae, genus Badnavirus. Fragments of two additional novel sequences resembling those found in members of the family Caulimoviridae were also identified in the black pepper sample, and the viruses from which they were derived were tentatively named Piper DNA virus 1 and 2.

Genome sequence of vanilla distortion mosaic virus infecting Coriandrum sativum.

The 9573-nucleotide genome of a potyvirus was sequenced from a Coriandrum sativum plant from India with viral symptoms. On analysis, this virus was shown to have greater than 85 % nucleotide sequence identity to vanilla distortion mosaic virus (VDMV). Analysis of the putative coat protein sequence confirmed that this virus was in fact VDMV, with greater than 91 % amino acid sequence identity. The genome appears to encode a 3083-amino-acid polyprotein potentially cleaved into the 10 mature proteins expected in potyviruses. Phylogenetic analysis confirmed that VDMV is a distinct but ungrouped member of the genus Potyvirus.

The complete genome sequences of two isolates of potato black ringspot virus and their relationship to other isolates and nepoviruses.

The complete nucleotide sequences of RNA 1 and RNA 2 of the nepovirus potato black ringspot virus (PBRSV) from two different isolates were determined, as well as partial sequences from two additional isolates. RNA1 is 7,579-7,598 nucleotides long and contains one single open reading frame (ORF), which is translated into a large polyprotein with 2,325 amino acids and a molecular weight of 257 kDa. The complete sequence of RNA2 ranges from 3857 to 3918 nt between the different isolates. It encodes a polyprotein of 1079-1082 amino acids with a molecular weight of 120 kDa. Sequence comparison using the Pro-Pol region and CP showed that all four isolates formed two distinct groups, corresponding to potato and arracacha, that were closely related to each other and also to tobacco ringspot virus (TRSV). Comparing our data to those obtained with other nepoviruses, our results confirm that PBRSV belongs to a distinct species and is a member of subgroup A in the genus Nepovirus based on its RNA2 size, genome organization, and nucleotide sequence.

Complete genome sequence of arracacha virus B: a novel cheravirus.

The complete genome sequences of RNA1 and RNA2 of the oca strain of the potato virus arracacha virus B were determined using next-generation sequencing. The RNA1 molecule is predicted to encode a 259-kDa polyprotein with homology to proteins of the cheraviruses apple latent spherical virus (ALSV) and cherry rasp leaf virus (CRLV). The RNA2 molecule is predicted to encode a 102-kDa polyprotein which also has homology to the corresponding protein of ALSV and, to a lesser degree, CRLV (30 % for RNA1, 24 % for RNA2). Detailed analysis of the genome sequence confirms that AVB is a distinct member of the genus Cheravirus.

The complete genome sequence of the Tanzanian strain of Cassava brown streak virus and comparison with the Ugandan strain sequence.

The complete genome sequence for an isolate of the Ugandan and Tanzanian strain types of Cassava brown streak virus have been determined using the novel approach of non-directed next generation sequencing. Comparison of the genome sequences revealed that CBSV is highly heterogeneous at the isolate level as well as the strain level. The isolate of the Ugandan strain was found to have a genome 9,070 nucleotides long coding for a polypeptide with 2,902 amino acid residues. The isolate of the Tanzanian strain was 9,008 nucleotides long and coded for a polypeptide with 2,916 amino acid residues. Nucleotide identity between the isolates across the genome was 76%, with protein encoding regions 57-77% and individual proteins had 65-91% amino acid similarity. In addition between the two strains four protein products (PIPO, CI, NIa-Vpg and coat protein) varied in size and an unusual HAM1-like protein, whilst of identical nucleotide length, was found to have the lowest homology. The implication of diversity of CBSV is discussed in the context of speciation, evolution, development of diagnostics, and breeding for resistance.

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.

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. METHODS AND RESULTS: A LAMP assay was designed based on the intergenic spacer of the B. cinerea nuclear ribosomal DNA (rDNA). The resulting assay was characterized in terms of sensitivity and specificity using DNA extracted from cultures. The assay consistently amplified 65 pg B. cinerea DNA. No cross-reactivity was observed with a range of other fungal pathogens, with the exception of the closely related species Botrytis pelargonii. Use of a novel real-time LAMP platform (the OptiGene Genie I) allowed detection of B. cinerea in infected rose petals, with amplification occurring in <15 min. CONCLUSIONS: The LAMP assay that was developed is suitable for rapid detection of B. cinerea in infected plant material. SIGNIFICANCE AND IMPACT OF THE STUDY: The LAMP method combines the sensitivity and specificity of nucleic acid-based methods with simplified equipment and a reduced reaction time. These features make the method potentially suitable for on-site use, where the results of testing could help to inform decisions regarding the storage and processing of commodities affected by B. cinerea, such as cut flowers, fruit and vegetables.

Development of a real-time PCR assay for the detection of Trichinella spiralis in situ.

Trichinellosis is a widespread zoonotic disease caused by nematodes of the genus Trichinella. Most human infections are caused by Trichinella spiralis, with pig meat being the main source of infection. As a consequence, all countries in the EU inspect slaughtered animals to prevent the distribution of infected meat to consumers. However, Trichinella spp. infect nearly all orders of mammals and so wildlife monitoring is often required in regions that want to provide evidence of negligible risk of infection in pigs. Surveys of the parasite in the Red fox are generally accepted as evidence of the wildlife reservoir. The EU reference method of detection in food animals for human consumption involves digestion of the host muscle followed by microscopic screening of the resultant sediment for trichinae and the method has been adapted for use with foxes. This work describes the development of a real-time PCR assay for the detection of Trichinella in fox tissue. The assay was designed to the Internal Transcribed Spacer 2 region of the Trichinella genome. Initial assay development was carried out using infected mouse tissue, as positive foxes have not been reported in the UK since 1957. The developed assay, which was shown to be specific for T. spiralis, was then tested using fox muscle spiked with isolated larvae at the rate of 1 larva per gram (LPG) of muscle tissue, as this is the theoretical detection limit using the digest method, as well as 0.5 and 0.1 LPG. The PCR assay was shown to detect the larvae at the higher infection rates and, by testing dilutions of the extracted DNA, it was demonstrated that a potential limit of detection of approx. 0.01 larvae per gram of tissue homogenate may be possible.

Single-step RT real-time PCR for sensitive detection and discrimination of Potato virus Y isolates.

Potato virus Y (PVY) has a worldwide distribution and infects several economically important crops from the Solanaceae family. The emergence and spread of the PVYNTN strain, which is the causative agent of potato tuber necrotic ringspot disease (PTNRD), has lead to large economic losses and highlighted the need for accurate discrimination of the different PVY strains. Detection and differentiation of PVY isolates is mainly based on a combination of ELISA, RT-PCR and bioassays; however, PVYNTN isolates are particularly difficult to differentiate from standard PVYN without the use of time-consuming bioassays. A strong correlation has been identified previously between the ability to induce PTNRD and the presence of a recombination point in the virus coat protein. An RT real-time PCR assay has been developed to enable detection of isolates with the recombination point, therefore, enabling rapid differentiation between potentially tuber necrotic PVYNTN isolates and standard PVYN isolates. The assay is also able to detect the presence of PVYO isolates. To aid with routine testing, immuno-capture and post-ELISA virus release were introduced; when coupled with RT real-time PCR the sensitivity of the assays were up to seven orders of magnitude higher than ELISA. The assay was shown to be a suitable method for rapid large-scale diagnostic testing of PVY in different types of plant material including tubers, and specific screening for potentially tuber necrotic recombinant isolates.

Development of real-time RT-PCR assays for the detection of Cucumber vein yellowing virus (CVYV) and Cucurbit yellow stunting disorder virus (CYSDV) in the whitefly vector Bemisia tabaci.

Reverse transcription followed by real-time PCR assays based on TaqMan chemistry have been developed for the detection and quantification of Cucumber vein yellowing virus (CVYV) and Cucurbit yellow stunting disorder virus (CYSDV) in individual adults of the whitefly vector Bemisia tabaci. The method includes an internal control for the detection of a gene from B. tabaci to compensate for variations in extraction efficiency. The assays designed were used to estimate proportions of viruliferous whiteflies collected from commercial greenhouse-grown crops in Spain. In a significant number of whiteflies, both viruses were detected and their amounts were estimated. The assays could be used to assist risk assessment of CVYV and CYSDV which constitute limiting factors in cucurbit crops. They are also suited to investigating the epidemiology and plant-virus-vector relationships in these diseases.

The effects of surface structure mutations in Arabidopsis thaliana on the polarization of reflections from virus-infected leaves.

The way in which light is polarized when reflected from leaves can be affected by infection with plant viruses. This has the potential to influence viral transmission by insect vectors due to altered visual attractiveness of infected plants. The optical and topological properties of cuticular waxes and trichomes are important determinants of how light is polarized upon reflection. Changes in expression of genes involved in the formation of surface structures have also been reported following viral infection. This paper investigates the role of altered surface structures in virus-induced changes to polarization reflection from leaves. The percentage polarization of reflections from Arabidopsis thaliana cer5, cer6 and cer8 wax synthesis mutants, and the gl1 leaf hair mutant, was compared to those from wild-type (WT) leaves. The cer5 mutant leaves were less polarizing than WT on the adaxial and abaxial surfaces; gl1 leaves were more polarizing than WT on the adaxial surfaces. The cer6 and cer8 mutations did not significantly affect polarization reflection. The impacts of Turnip vein clearing virus (TVCV) infection on the polarization of reflected light were significantly affected by cer5 mutation, with the reflections from cer5 mutants being higher than those from WT leaves, suggesting that changes in CER5 expression following infection could influence the polarization of the reflections. There was, however, no significant effect of the gl1 mutation on polarization following TVCV infection. The cer5 and gl1 mutations did not affect the changes in polarization following Cucumber mosaic virus (CMV) infection. The accumulation of TVCV and CMV did not differ significantly between mutant and WT leaves, suggesting that altered expression of surface structure genes does not significantly affect viral titres, raising the possibility that if such regulatory changes have any adaptive value it may possibly be through impacts on viral transmission.

Development of real-time and conventional RT-PCR assays for the detection of potato yellow vein virus (PYVV).

Potato yellow vein virus (PYVV) is considered a quarantine pathogen in the European and Mediterranean Plant Protection Organization (EPPO) area. This virus is widespread and damaging at its centre of origin in South America. Current detection methods are either time-consuming or difficult to interpret. This paper reports the development of a sensitive, high throughput, real-time reverse transcription (RT)-PCR assay, based on TaqMan chemistry, suitable for PYVV detection. In addition, a reliable conventional RT-PCR assay for PYVV detection is also presented. Although less sensitive (1000 times less sensitive in direct comparison), this method requires less sophisticated equipment and as such should be a useful alternative to the real-time technique in some testing laboratories. The two assays presented here could assist in the implementation of quarantine measures for PYVV identification and in routine indexing of PYVV for the production of virus-free seed potatoes in areas of South America where the virus is highly damaging.

Fourier transform infra-red spectroscopy using an attenuated total reflection probe to distinguish between Japanese larch, pine and citrus plants in healthy and diseased states.

FTIR spectroscopy coupled with an Attenuated Total Reflection (ATR) sampling probe has been demonstrated as a technique for detecting disease in plants. Spectral differences were detected in Japanese Larch (Larix kaempferi) infected with Phytophthora ramorum at 3403cm(-1) and 1730cm(-1), from pine (Pinus spp.) infected with Bursaphelenchus xylophilus at 1070cm(-1), 1425cm(-)1, 1621cm(-1) and 3403cm(-1) and from citrus (Citrus spp.) infected with 'Candidatus liberibacter' at 960cm(-1), 1087cm(-1), 1109cm(-1), 1154cm(-1), 1225cm(-1), 1385cm(-1), 1462cm(-1), 1707cm(-1), 2882cm(-1), 2982cm(-1) and 3650cm(-1). A spectral marker in healthy citrus has been identified as Pentanone but is absent from the diseased sample spectra. This agrees with recent work by Aksenov, 2014. Additionally, the spectral signature of Cutin was identified in the spectra of Pinus spp. and Citrus spp. and is consistent with work by Dubis, 1999 and Heredia-Guerrero, 2014.

Investigating the specificity of real-time PCR assays using synthetic oligonucleotides.

Potato spindle tuber viroid (PSTVd) causes damaging diseases of solanaceous crops and is a quarantine pathogen in the European Union. Previously a one-tube real-time RT-PCR assay based on TaqMan chemistry was developed and shown to be ideally suited to PSTVd detection. However, since it was impossible to trace infected plant material for every published PSTVd sequence reported, in silico predictions were made about assay specificity based on the positions of nucleotide polymorphisms within the published viroid sequences and the regions of the primers and probe. The predictions could not be verified due to the absence of viroid material. This paper describes work investigating the detection of these sequence variants by designing synthetic oligonucleotides to sequences from the database and testing them with a real-time PCR assay. The results show that all PSTVd sequence variants are detected, and that the closely related Mexican papita viroid is also detected, although with a lower efficiency. The paper gives indications as to what effect nucleotide changes at different positions within primers and probes might do and should aid in the testing of future assays, although it is difficult to draw fixed rules about the possible effect changes may have.

The use of real-time RT-PCR (TaqMan) and post-ELISA virus release for the detection of Beet necrotic yellow vein virus types containing RNA 5 and its comparison with conventional RT-PCR.

Real-time RT-PCR (TaqMan) assays were developed for the specific detection of Beet necrotic yellow vein virus (BNYVV). The two assays designed were a broad-spectrum one that detected RNA 2 from all types and a second designed to detect types containing RNA 5. The assays were validated against a range of different isolates from Europe and the Far East. These real-time assays were compared to a conventional RT-PCR assay for the detection of RNA 5. Sensitivity comparisons showed that for the detection of RNA 5, TaqMan was 10,000 times more sensitive than the conventional RT-PCR assay. Further improvements were made to the test procedure by using post-ELISA virus release (VR), as an alternative to RNA extraction. This significantly increased the speed of processing samples and reduced the staff input required, allowing the TaqMan assay to be used routinely as part of an annual survey of UK field samples.

Use of quantitative molecular diagnostic assays to investigate fusarium dry rot in potato stocks and soil.

ABSTRACT Specific and sensitive quantitative diagnostics, based on real-time (TaqMan) polymerase chain reaction (PCR) and PCR enzyme-linked immunosorbent assay, were developed to detect dry-rot-causing Fusarium spp. (F. avenaceum, F. coeruleum, F. culmorum, and F. sulphureum). Each assay detected Fusarium spp. on potato seed stocks with equal efficiency. Four potato stocks, sampled over two seed generations from Scottish stores, were contaminated with F. avenaceum, F. sulphureum, F. culmorum, F. coeruleum or a combination of species, and there was a general trend towards increased Fusarium spp. contamination in the second generation of seed sampled. F. sulphureum and F. coeruleum caused significantly (P < 0.05) more disease in storage than the other species when disease-free tubers of potato cvs. Spunta and Morene were inoculated at a range of inoculum concentrations (0, 10(4), 10(5), and 10(6) conidia/ml). Increased DNA levels were correlated with increased disease severity between 8 and 12 weeks of storage. The threshold inoculum levels resulting in significant disease development on both cultivars were estimated to be 10(4) conidia/ml for F. sulphureum and 10(5) conidia/ml for F. coeruleum. To study the effect of soil infestation and harvest date on disease incidence, seed tubers of cvs. Morene and Spunta were planted in a field plot artificially infested with the four Fusarium spp. F. culmorum and F. sulphureum were detected in soil taken from these plots at harvest, and F. sulphureum DNA levels increased significantly (P < 0.05) at the final harvest. All four Fusarium spp. were detected in progeny tubers. There was a trend toward higher levels of F. culmorum detected in progeny tubers at the earliest harvest date, and higher levels of F. sulphureum at the final harvest. The use of diagnostic assays to detect fungal storage rot pathogens and implications for disease control strategies are discussed.

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.

Development of a lateral flow device for in-field detection and evaluation of PCR-based diagnostic methods for Xanthomonas campestris pv. musacearum, the causal agent of banana xanthomonas wilt.

Xanthomonas campestris pv. musacearum (Xcm) is the causal agent of banana xanthomonas wilt, a major threat to banana production in eastern and central Africa. The pathogen is present in very high levels within infected plants and can be transmitted by a broad range of mechanisms; therefore early specific detection is vital for effective disease management. In this study, a polyclonal antibody (pAb) was developed and deployed in a lateral flow device (LFD) format to allow rapid in-field detection of Xcm. Published Xcm PCR assays were also independently assessed: only two assays gave specific amplification of Xcm, whilst others cross-reacted with non-target Xanthomonas species. Pure cultures of Xcm were used to immunize a rabbit, the IgG antibodies purified from the serum and the resulting polyclonal antibodies tested using ELISA and LFD. Testing against a wide range of bacterial species showed the pAb detected all strains of Xcm, representing isolates from seven countries and the known genetic diversity of Xcm. The pAb also detected the closely related Xanthomonas axonopodis pv. vasculorum (Xav), primarily a sugarcane pathogen. Detection was successful in both naturally and experimentally infected banana plants, and the LFD limit of detection was 105 cells mL-1. Whilst the pAb is not fully specific for Xcm, Xav has never been found in banana. Therefore the LFD can be used as a first-line screening tool to detect Xcm in the field. Testing by LFD requires no equipment, can be performed by non-scientists and is cost-effective. Therefore this LFD provides a vital tool to aid in the management and control of Xcm.