[Show abstract][Hide abstract]ABSTRACT: Alteration of leaf surface phenotypes due to virus infection has the potential to affect the likelihood of colonisation by insect vectors, or to affect their feeding activities. The aim of this study was to investigate whether viruses that rely on insects for their transmission, and which can be sensitive to the polarization of light, affect the percentage polarization of light reflected from leaves. We also set out to discover whether a correlation exists between the expression of ECERIFERUM (CER) genes involved in cuticular wax synthesis and the polarization of the light reflected from the leaf surfaces. It was found that the aphid-vectored viruses Potato virus Y and Cucumber mosaic virus (CMV) caused significant reductions in the percentage polarization of light reflected from the abaxial surfaces of leaves of Nicotiana tabacum, whereas the non-insect-vectored viruses Tobacco mosaic virus and Pepino mosaic virus did not induce this effect. In Arabidopsis thaliana, there was little difference in the impacts of CMV and the non-insect-vectored Turnip vein clearing virus on polarization reflection, with both viruses increasing the percentage polarization of light reflected from the abaxial surfaces of leaves. There was a trend towards increased accumulation of CER6 transcripts in N. tabacum and A. thaliana when infected with aphid-vectored viruses. No significant effect of infection on trichome densities was found in A. thaliana, suggesting that alterations to the formation of cuticular waxes may be the more likely phenotypic change on the leaf surface contributing to the changes in polarization reflection. The possible impacts and adaptive significance of these effects with regard to viral transmission by insects are discussed.
[Show abstract][Hide abstract]ABSTRACT: Potato is the fourth most important crop worldwide that is used as a staple food, after rice, wheat and maize. The crop can be affected by a large number of pathogens, including fungi, oomycetes, bacteria and viruses. Diseases caused by viruses are among the most important factors contributing to reduced quality and yield of the crop. Potato mop-top virus (genus Pomovirus) induces necrotic flecks in the tuber flesh and skin of potato in temperate countries. Spongospora subterranea is the vector of PMTV. Both the virus and its vector cause disease in potato. In Colombia, PMTV has been detected throughout the country together with a novel pomo-like virus in the centre (Cundinamarca and Boyacá) and south west (Nariño) of the country. We studied the molecular and biological characteristics of this novel virus. Its genome resembles those of members of the genus Pomovirus, and it is closely related to PMTV. It induces mild systemic symptoms in Nicotiana benthamiana (mosaic, branch curling), but no symptoms in N. tabacum, N. debneyi and Chenopodium amaranticolor. The proposed name for the virus is "Colombian potato soil-borne virus" (CPSbV). Additionally, another pomo-like virus was identified in Nariño. This virus induces severe systemic stem declining and mild mosaic in N. benthamiana. The tentative name "soil-borne virus 2" (SbV2) is proposed for this virus. No vectors have been identified for these viruses despite several attempts. This work focused on the characterisation of CPSbV. The risk posed by these viruses if they are introduced into new territories is discussed.
No preview · Article · Mar 2016 · Archives of Virology
[Show abstract][Hide abstract]ABSTRACT: Potato mop-top virus (PMTV) causes necrotic flecks inside and on tubers in temperate countries. In South America, these symptoms have not been observed, although the presence of the virus has been confirmed in the Andes and in Central America. To characterize PMTV isolates from the Andes, soil samples were taken from the main potato-producing regions in Colombia and virus was recovered by planting Nicotiana benthamiana as bait plants. The complete genomes of five isolates were sequenced and three of the isolates were inoculated to four different indicator plants. Based on sequence comparisons, three types of RNA-CP (RNA2) and RNA-TGB (RNA3) were found. The isolates from the centre of the country (CO3 and CO4) were similar to isolates from Europe. The genomes of CO1, CO2 and CO5 differ from other PMTV isolates, placing them in a separate clade in phylogenetic trees. The three Colombian isolates (CO1, CO2 and CO5) only induced slightly different symptoms in the indicator plants. However, the isolate from the northwest of the country (CO1) induced stronger symptoms in N. benthamiana including severe stunting. A correlation between the genotype of the isolates and the symptoms they induced on indicator plants was not found.
[Show abstract][Hide abstract]ABSTRACT: 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 X. vasicola, seven other bacterial pathogens/endophytes of Musa isolates 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 minutes from bacterial cultures. Symptomatic field samples of Musa from Uganda were tested and all produced amplification in less than 13 minutes.
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. This article is protected by copyright. All rights reserved.
No preview · Article · Oct 2015 · Journal of Applied Microbiology
[Show abstract][Hide abstract]ABSTRACT: Diagnostic microarrays are a useful tool for the simultaneous detection of multiple targets. In this chapter we describe the use of a simple tube-based microarray platform for the detection of plant infecting viruses and viroids.
No preview · Article · May 2015 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract]ABSTRACT: Chalara fraxinea is the causal agent of ash dieback, a disease affecting Fraxinus excelsior and F. angustifolia across Europe. Loop-mediated isothermal amplification (LAMP) is a rapid, DNA-based method which can be used for specific detection of plant pathogens in infected material. The combination of a rapid LAMP assay for C. fraxinea with a simple sample preparation method in a user-friendly kit format raises the potential for testing to be carried out away from conventional laboratory facilities, to expedite measure to manage this damaging disease.
No preview · Article · May 2015 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract]ABSTRACT: Pepino mosaic virus (PepMV) poses a worldwide threat to the tomato industry. Considerable differences at the genetic level allow for the distinction of four main genotypic clusters; however, the basis of the phenotypic outcome is difficult to elucidate. This work reports the generation of wild-type PepMV infectious clones of both EU (mild) and CH2 (aggressive) genotypes, from which chimeric infectious clones were created. Phenotypic analysis in three solanaceous hosts, Nicotiana benthamiana, Datura stramonium and Solanum lycopersicum, indicated that a PepMV pathogenicity determinant mapped to the 3′-terminal region of the genome. Increased aggression was only observed in N. benthamiana, showing that this factor is host specific. The determinant was localized to amino acids 11–26 of the N-terminal coat protein (CP) region; this is the first report of this region functioning as a virulence factor in PepMV.
[Show abstract][Hide abstract]ABSTRACT: The potato Rx gene provides resistance against Pepino mosaic virus (PepMV) in tomato; however, recent work has suggested that the resistance conferred may not be durable. Resistance breaking can probably be attributed to multiple mutations observed to accumulate in the capsid protein (CP) region of resistance-breaking isolates, but this has not been confirmed through directed manipulation of an infectious PepMV clone. The present work describes the introduction of two specific mutations, A-T78 and A-T114, into the coat protein minimal elicitor region of an Rx-controlled PepMV isolate of the EU genotype. Enzyme-linked immunosorbent assay (ELISA) and phenotypic evaluation were conducted in three Rx-expressing and wild-type solanaceous hosts: Nicotiana benthamiana, Nicotiana tabacum and Solanum lycopersicum. Mutation A-T78 alone was sufficient to confer Rx-breaking activity in N. benthamiana and S. lycopersicum, whereas mutation A-T114 was found to be associated, in most cases, with a secondary A-D100 mutation to break Rx-mediated resistance in S. lycopersicum. These results suggest that the need for a second, fitness-restoring mutation may be dependent on the PepMV mutant under consideration. Both mutations conferred Rx breaking in S. lycopersicum, whereas neither conferred Rx breaking in N. tabacum and only A-T78 allowed Rx breaking in N. benthamiana, suggesting that Rx may function in a different manner depending on the genetic background in which it is present.
No preview · Article · Mar 2015 · Molecular Plant Pathology
[Show abstract][Hide abstract]ABSTRACT: The ability to provide a fast, inexpensive and reliable diagnostic for any given viral infection is a key parameter in efforts to fight and control these ubiquitous pathogens. The recent developments of high-throughput sequencing (also called Next Generation Sequencing - NGS) technologies and bioinformatics have drastically changed the research on viral pathogens. It is now raising a growing interest for virus diagnostics. This review provides a snapshot vision on the current use and impact of high throughput sequencing approaches in plant virus characterization. More specifically, this presentation highlights the potential of these new technologies and their interplay with current protocols in the future of molecular diagnostic of plant viruses. The current limitations that will need to be addressed for a wider adoption of high-throughput sequencing in plant virus diagnostics are thoroughly discussed. This paradigm change gave rise to the COST Action 1407 which is currently launched. This Action, its objectives and expected impacts will be presented.
[Show abstract][Hide abstract]ABSTRACT: Internal necrosis of carrot has been observed in UK carrots for at least 10 years, and has been anecdotally linked to virus infection. In the 2009 growing season some growers had up to 10% of yield with these symptoms. Traditional diagnostic methods are targeted towards specific pathogens. By using a metagenomic approach with high throughput sequencing technology, other, as yet unidentified causes of root necrosis were investigated. Additionally a statistical analysis has shown which viruses are most closely associated with disease symptoms. Carrot samples were collected from a crop exhibiting root necrosis (102 Affected: 99 Unaffected) and tested for the presence of the established carrot viruses: Carrot red leaf virus (CtRLV), Carrot mottle virus (CMoV), Carrot red leaf associated viral RNA (CtRLVaRNA) and Parsnip yellow fleck virus (PYFV). The presence of these viruses was not associated with symptomatic carrot roots either as single viruses or in combinations. A sub-sample of carrots of mixed symptom status was subjected to MiSeq sequencing. The results from these tests suggested Carrot yellow leaf virus (CYLV) was associated with symptomatic roots. Additionally a novel Torradovirus, a novel Closterovirus and two novel Betaflexiviradae related plant viruses were detected. A specific diagnostic test was designed for CYLV. Of the 102 affected carrots, 98% were positive for CYLV compared to 22% of the unaffected carrots. From these data we conclude that although we have yet to practically demonstrate a causal link, CYLV appears to be strongly associated with the presence of necrosis of carrots.
[Show abstract][Hide abstract]ABSTRACT: 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.
No preview · Article · Sep 2014 · Archives of Virology
[Show abstract][Hide abstract]ABSTRACT: Xanthomonas campestris pathovar 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 we have developed a polyclonal antibody (pAb) and deployed this in a lateral flow device (LFD) format to allow rapid in-field detection of Xcm. We also independently assessed published Xcm PCR assays: only two assays gave specific amplification of Xcm, whilst others cross-reacted with non-target Xanthomonas species. Pure cultures of Xcm were used to immunise 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 pathovar 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. 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.This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract]ABSTRACT: Maize lethal necrosis (MLN) is a disease of maize caused by the combination of Maize chlorotic mottle virus (MCMV) and a potyvirus (Uyemote et al., 1981). MLN caused by MCMV and the potyvirus Sugarcane mosaic virus (SCMV) was identified in Kenya in 2012 (Adams et al., 2013) and associated with significant damage. Subsequent to the outbreak in Kenya, MLN symptoms have been associated with MCMV and SCMV in Tanzania and Uganda and, based on our initial findings, MCMV but not SCMV in Rwanda (ASARECA, 2013). The control of MLN is a priority for the East Africa region. We report here a fuller investigation of the causal agents of MLN from Rwanda.
In March and June 2013 samples of maize showing signs of MLN symptoms were received from the Northern Province of Rwanda for MCMV and SCMV testing. The samples were tested by real-time PCR as described in Adams et al. (2013). By this method all samples tested positive for MCMV and negative for SCMV. In order to determine if MCMV was responsible for the symptoms, or if another potyvirus was present, deep sequencing of total RNA extracted from four of the maize samples was performed. Indexed ScriptSeq libraries (Epibio, UK) were produced and sequenced on a MiSeq sequencer (Illumina, UK) using 500 cycle V2 reagents. The resulting 10 million 250 bp paired-end reads were split by index, quality filtered to a score of above Q20 and assembled using Trinity (Grabherr et al., 2011). Contigs over 200 bp were then compared to the GenBank nr database using BLASTX and the resulting data analysed using MEGAN as previously reported to generate draft genomes (Adams et al., 2013).
Complete genomes of MCMV were recovered from all four maize samples from Rwanda (GenBank Accession Nos. KF744393-KF744396) and shown to have a very close relatedness with MCMV from Kenya and China (99% homology), and with some separation from MCMV from the United States (97-96% homology) (Fig. 1). Complete SCMV genomes were found in three of the four samples (KF744390- KF744392). Fig. 2 shows the relationship between these and previous isolates. The SCMV isolated from Rwandan samples is distinct from that isolated in Kenya (87% identity) and most closely related (95% identity) to strain SCMV-DMB (BA00797). A closer examination of the sequence at the primer binding regions of the respective SCMV genomes shows a high degree of divergence and explain the negative results following real-time PCR testing using the assay designed to the SCMV sequence from Kenya (Adams et al., 2013).
These data would support a shared origin for MCMV in Rwanda and Kenya, but different sources for SCMV. This is not unexpected as SCMV has been observed for many years in East Africa (Louie, 1980), whereas MCMV is believed to be new to the region (Adams et al., 2013). MCMV would now appear to be spreading across the East Africa region, potentially in seed, and causative of MLN where populations of SCMV are already endemic. These findings have implications for the spread of MLN throughout Africa and for the testing for MCMV and SCMV. Control of the disease may prove more effective by initiating quarantine measures to counter the movement of MCMV, rather than attempting to control the endemic SCMV. This report confirms that MLN, caused by MCMV and SCMV, is now present in Rwanda and that this is most probably due to the recent introduction of MCMV.
[Show abstract][Hide abstract]ABSTRACT: 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 FD LAMP assay was shown to be specific and extremely sensitive, since 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 optimised for on-site detection and can be completed in one hour. The homogenisation procedure of the grapevine samples (leaf vein, flower or berry) was optimised to allow direct testing of the crude homogenates with the LAMP assay, without the need for a DNA extraction, and was shown to be extremely sensitive.This article is protected by copyright. All rights reserved.