Innovative tools for detection of plant pathogenic viruses and bacteria

Detection of harmful viruses and bacteria in plant material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. The techniques available have evolved significantly in the last few years to achieve rapid and reliable detection of pathogens, extraction of the target from the sample being important for optimising detection. For viruses, sample preparation has been simplified by imprinting or squashing plant material or insect vectors onto membranes. To improve the sensitivity of techniques for bacterial detection, a prior enrichment step in liquid or solid medium is advised. Serological and molecular techniques are currently the most appropriate when high numbers of samples need to be analysed. Specific monoclonal and/or recombinant antibodies are available for many plant pathogens and have contributed to the specificity of serological detection. Molecular detection can be optimised through the automatic purification of nucleic acids from pathogens by columns or robotics. New variants of PCR, such as simple or multiplex nested PCR in a single closed tube, co-operative- PCR and real-time monitoring of amplicons or quantitative PCR, allow high sensitivity in the detection of one or several pathogens in a single assay. The latest development in the analysis of nucleic acids is microarray technology, but it requires generic DNA/RNA extraction and pre-amplification methods to increase detection sensitivity. The advances in research that will result from the sequencing of many plant pathogen genomes, especially now in the era of proteomics, represent a new source of information for the future development of sensitive and specific detection techniques for these microorganisms

Genetic diversity reflects geographical origin of Ralstonia solanacearum strains isolated from plant and water sources in Spain

The characterization and intraspecific diversity of a collection of 45 Ralstonia solanacearum strains isolated in Spain from different sources and geographical origins is reported. To test the influence of the site and the host on strain diversity, phenotypic and genotypic analysis were performed by a polyphasic approach. Biochemical and metabolic profiles were compared. Serological relationship was evaluated by Indirect-ELISA using polyclonal and monoclonal antibodies. For genotypic analysis, hrpB and egl DNA sequence analysis, repetitive sequences (rep-PCR), amplified fragment length polymorphism (AFLP) profiles and macrorestriction with XbaI followed by pulsed field gel electrophoresis (PFGE) were performed.The biochemical and metabolic characterization, serological tests, rep-PCR typing and phylogenetic analysis showed that all analysed strains belonged to phylotype II sequevar 1 and shared homogeneous profiles. However, interesting differences among strains were found by AFLP and macrorestriction with XbaI followed by PFGE techniques, some profiles being related to the geographical origin of the strains. Diversity results obtained offer new insights into the biogeography of this quarantine organism and its possible sources and reservoirs in Spain and Mediterranean countries.Keywords: Bacterial wilt · potato · soil · PFGE · AFL

Lateral flow immunoassay for on-site detection of Xanthomonas arboricola pv. Pruni in symptomatic field samples

[EN] Xanthomonas arboricola pv. pruni is a quarantine pathogen and the causal agent of the bacterial spot disease of stone fruits and almond, a major threat to Prunus species. Rapid and specific detection methods are essential to improve disease management, and therefore a prototype of a lateral flow immunoassay (LFIA) was designed for the detection of X. arboricola pv. pruni in symptomatic field samples. It was developed by producing polyclonal antibodies which were then combined with carbon nanoparticles and assembled on nitrocellulose strips. The specificity of the LFIA was tested against 87 X. arboricola pv. pruni strains from different countries worldwide, 47 strains of other Xanthomonas species and 14 strains representing other bacterial genera. All X. arboricola pv. pruni strains were detected and cross-reactions were observed only with four strains of X. arboricola pv. corylina, a hazelnut pathogen that does not share habitat with X. arboricola pv. pruni. The sensitivity of the LFIA was assessed with suspensions from pure cultures of three X. arboricola pv. pruni strains and with spiked leaf extracts prepared from four hosts inoculated with this pathogen (almond, apricot, Japanese plum and peach). The limit of detection observed with both pure cultures and spiked samples was 10(4) CFU ml(-1). To demonstrate the accuracy of the test, 205 samples naturally infected with X. arboricola pv. pruni and 113 samples collected from healthy plants of several different Prunus species were analyzed with the LFIA. Results were compared with those obtained by plate isolation and real time PCR and a high correlation was found among techniques. Therefore, we propose this LFIA as a screening tool that allows a rapid and reliable diagnosis of X. arboricola pv. pruni in symptomatic plants.The work was supported by the following: Instituto Nacional de Tecnologia Agraria y Alimentaria, Project RTA-2011-00140-C03-01 (http://www.inia.es), PLS MTG EMN MML; Instituto Nacional de Tecnologia Agraria y Alimentaria, FPI-INIA grant (http://www.inia.es), PLS; Generalitat Valenciana (Prometeo II 2014/040) (http://www.gva.es), PN RP AM; Ministerio de Economia y Competitividad (MINECO) (CTQ2013-45875R) (http://www.mineco.gob.es), PN RP AM; European Social Fund, PLS MTG EMN MML; and European Regional Development Fund, PLS MTG EMN MML.López-Soriano, P.; Noguera Murray, PS.; Gorris, MT.; Puchades, R.; Maquieira Catala, A.; Marco-Noales, E.; López, M. (2017). Lateral flow immunoassay for on-site detection of Xanthomonas arboricola pv. Pruni in symptomatic field samples. PLoS ONE. 12(4):1-13. https://doi.org/10.1371/journal.pone.0176201113124Tjou-Tam-Sin, N. N. A., van de Bilt, J. L. J., Bergsma-Vlami, M., Koenraadt, H., Naktuinbouw, J. W., van Doorn, J., … Martin, W. S. (2012). First Report of Xanthomonas arboricola pv. pruni in Ornamental Prunus laurocerasus in the Netherlands. Plant Disease, 96(5), 759-759. doi:10.1094/pdis-04-11-0265-pdnPothier, J. F., Vorhölter, F.-J., Blom, J., Goesmann, A., Pühler, A., Smits, T. H. M., & Duffy, B. (2011). The ubiquitous plasmid pXap41 in the invasive phytopathogen Xanthomonas arboricola pv. pruni: complete sequence and comparative genomic analysis. FEMS Microbiology Letters, 323(1), 52-60. doi:10.1111/j.1574-6968.2011.02352.xPalacio-Bielsa, A., Cubero, J., Cambra, M. A., Collados, R., Berruete, I. M., & López, M. M. (2010). Development of an Efficient Real-Time Quantitative PCR Protocol for Detection ofXanthomonas arboricolapv. pruni inPrunusSpecies. Applied and Environmental Microbiology, 77(1), 89-97. doi:10.1128/aem.01593-10Xanthomonas arboricola pv. pruni. (2006). EPPO Bulletin, 36(1), 129-133. doi:10.1111/j.1365-2338.2006.00925.xPagani MC. An ABC transporter protein and molecular diagnoses of Xanthomonas arboricola pv. pruni causing bacterial spot of stone fruits. Raleigh, North Carolina, USA: North Carolina State University, PhD thesis. 2004; Online, http://repository.lib.ncsu.edu/ir/bitstream/1840.16/4540/1/etd.pdfPark, S. Y., Lee, Y. S., Koh, Y. J., Hur, J.-S., & Jung, J. S. (2010). Detection of Xanthomonas arboricola pv. pruni by PCR using primers based on DNA sequences related to the hrp genes. The Journal of Microbiology, 48(5), 554-558. doi:10.1007/s12275-010-0072-3Pothier, J. F., Pagani, M. C., Pelludat, C., Ritchie, D. F., & Duffy, B. (2011). A duplex-PCR method for species- and pathovar-level identification and detection of the quarantine plant pathogen Xanthomonas arboricola pv. pruni. Journal of Microbiological Methods, 86(1), 16-24. doi:10.1016/j.mimet.2011.03.019Ballard, E. L., Dietzgen, R. G., Sly, L. I., Gouk, C., Horlock, C., & Fegan, M. (2011). Development of a Bio-PCR Protocol for the Detection of Xanthomonas arboricola pv. pruni. Plant Disease, 95(9), 1109-1115. doi:10.1094/pdis-09-10-0650Boonham, N., Glover, R., Tomlinson, J., & Mumford, R. (2008). Exploiting generic platform technologies for the detection and identification of plant pathogens. European Journal of Plant Pathology, 121(3), 355-363. doi:10.1007/s10658-008-9284-3Posthuma-Trumpie, G. A., Korf, J., & van Amerongen, A. (2008). Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey. Analytical and Bioanalytical Chemistry, 393(2), 569-582. doi:10.1007/s00216-008-2287-2De Boer, S. H., & López, M. M. (2012). New Grower-Friendly Methods for Plant Pathogen Monitoring. Annual Review of Phytopathology, 50(1), 197-218. doi:10.1146/annurev-phyto-081211-172942Thornton, C. R., Groenhof, A. C., Forrest, R., & Lamotte, R. (2004). A One-Step, Immunochromatographic Lateral Flow Device Specific to Rhizoctonia solani and Certain Related Species, and Its Use to Detect and Quantify R. solani in Soil. Phytopathology®, 94(3), 280-288. doi:10.1094/phyto.2004.94.3.280Lane, C. R., Hobden, E., Walker, L., Barton, V. C., Inman, A. J., Hughes, K. J. D., … Barker, I. (2007). Evaluation of a rapid diagnostic field test kit for identification of Phytophthora species, including P. ramorum and P. kernoviae at the point of inspection. Plant Pathology, 56(5), 828-835. doi:10.1111/j.1365-3059.2007.01615.xSafenkova, I., Zherdev, A., & Dzantiev, B. (2012). Factors influencing the detection limit of the lateral-flow sandwich immunoassay: a case study with potato virus X. Analytical and Bioanalytical Chemistry, 403(6), 1595-1605. doi:10.1007/s00216-012-5985-8Safenkova, I. V., Pankratova, G. K., Zaitsev, I. A., Varitsev, Y. A., Vengerov, Y. Y., Zherdev, A. V., & Dzantiev, B. B. (2016). Multiarray on a test strip (MATS): rapid multiplex immunodetection of priority potato pathogens. Analytical and Bioanalytical Chemistry, 408(22), 6009-6017. doi:10.1007/s00216-016-9463-6Braun-Kiewnick, A., Altenbach, D., Oberhänsli, T., Bitterlin, W., & Duffy, B. (2011). A rapid lateral-flow immunoassay for phytosanitary detection of Erwinia amylovora and on-site fire blight diagnosis. Journal of Microbiological Methods, 87(1), 1-9. doi:10.1016/j.mimet.2011.06.015Safenkova, I. V., Zaitsev, I. A., Pankratova, G. K., Varitsev, Y. A., Zherdev, A. V., & Dzantiev, B. B. (2014). Lateral flow immunoassay for rapid detection of potato ring rot caused by Clavibacter michiganensis subsp. sepedonicus. Applied Biochemistry and Microbiology, 50(6), 675-682. doi:10.1134/s0003683814120011Hodgetts, J., Karamura, G., Johnson, G., Hall, J., Perkins, K., Beed, F., … Smith, J. (2014). Development of a lateral flow device for in-field detection and evaluation of PCR-based diagnostic methods forXanthomonas campestrispv.musacearum, the causal agent of banana xanthomonas wilt. Plant Pathology, 64(3), 559-567. doi:10.1111/ppa.12289Noguera, P., Posthuma-Trumpie, G. A., van Tuil, M., van der Wal, F. J., de Boer, A., Moers, A. P. H. A., & van Amerongen, A. (2010). Carbon nanoparticles in lateral flow methods to detect genes encoding virulence factors of Shiga toxin-producing Escherichia coli. Analytical and Bioanalytical Chemistry, 399(2), 831-838. doi:10.1007/s00216-010-4334-zCambra M, López MM. Titration of Agrobacterium radiobacter var. tumefaciens antibodies by using enzyme labeled anti-rabbit γ-globulines (ELISA indirect method). In: Proceedings of the 4th International Conference on Plant Pathogenic Bacteria, ed. Station Pathologie Végétale, INRA Angers, 1978. pp: 327–331.O’Keeffe, M., Crabbe, P., Salden, M., Wichers, J., Van Peteghem, C., Kohen, F., … Moneti, G. (2003). Preliminary evaluation of a lateral flow immunoassay device for screening urine samples for the presence of sulphamethazine. Journal of Immunological Methods, 278(1-2), 117-126. doi:10.1016/s0022-1759(03)00207-2PM 7/98 (2) Specific requirements for laboratories preparing accreditation for a plant pest diagnostic activity. (2014). EPPO Bulletin, 44(2), 117-147. doi:10.1111/epp.12118Lamichhane, J. R., & Varvaro, L. (2013). Xanthomonas arboricoladisease of hazelnut: current status and future perspectives for its management. Plant Pathology, 63(2), 243-254. doi:10.1111/ppa.12152Fischer-Le Saux, M., Bonneau, S., Essakhi, S., Manceau, C., & Jacques, M.-A. (2015). Aggressive Emerging Pathovars of Xanthomonas arboricola Represent Widespread Epidemic Clones Distinct from Poorly Pathogenic Strains, as Revealed by Multilocus Sequence Typing. Applied and Environmental Microbiology, 81(14), 4651-4668. doi:10.1128/aem.00050-15Bühlmann, A., Pothier, J. F., Tomlinson, J. A., Frey, J. 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Characterization of Monoclonal Antibodies Specific for Erwinia carotovora subsp. atroseptica and Comparison of Serological Methods for Its Sensitive Detection on Potato Tubers

Seven monoclonal antibodies (MAbs) to Erwinia carotovora subsp. atroseptica have been produced. One, called 4G4, reacted with high specificity for serogroup I of E. carotovora subsp. atroseptica, the most common serogroup on potato tubers in different serological assays. Eighty-six strains belonging to different E. carotovora subsp. atroseptica serogroups were assayed. Some strains of serogroup XXII also reacted positively. No cross-reactions were observed against other species of plant pathogenic bacteria or 162 saprophytic bacteria from potato tubers. Only one strain of E. chrysanthemi from potato cross-reacted. A comparison of several serological techniques to detect E. carotovora subsp. atroseptica on potato tubers was performed with MAb 4G4 or polyclonal antibodies. The organism was extracted directly from potato peels of artificially inoculated tubers by soaking or selective enrichment under anaerobiosis in a medium with polypectate. MAb 4G4 was able to detect specifically 240 E. carotovora subsp. atroseptica cells per ml by indirect immunofluorescence and immunofluorescence colony staining and after soaking by ELISA-DAS (double-antibody sandwich enzyme-linked immunosorbent assay) after enrichment. The same amount of cells was detected by using immunolectrotransfer with polyclonal antibodies, and E. carotovora subsp. atroseptica and subsp. carotovora were distinguished by the latter technique. ELISA-DAS using MAb 4G4 with an enrichment step also efficiently detected E. carotovora subsp. atroseptica in naturally infected tubers and plants

Detection and Diagnosis of Xylella fastidiosa by Specific Monoclonal Antibodies

Monoclonal antibodies (MAb) specific to Xylella fastidiosa were obtained through hybridoma technology using heat-treated somatic O antigens from LMG 17159strain. Ten stable hybrydoma clones secreting MAb were selected and their isotype was determined. The MAbs 2G1/PPD, IgG1 showed specificity for X. fastidiosa, detecting all the analyzed strains representing different subspecies, STs and hosts. Polyclonal antibodies (PAb) against X. fastidiosa were also produced and antiserum 17159-O/IVIA was selected for the highest titre and its excellent detection capability. MAb 2G1/PPD was tested against strain IVIA 5235 in PBS and in spiked raw extract samples from almond, olive, citrus, and other hosts and its sensitivity by DAS-ELISA was 104 CFU mL−1. The MAb also reacted with high affinity and avidity against X. fastidiosa by DASI-ELISA and Tissue print-ELISA. The diagnostic parameters of DAS-ELISA based on MAb were calculated and compared with the gold standard real-time PCR. The diagnostic specificity of MAb2G1/PPD was 100%, the diagnostic sensitivity was 88.5% compared to Harper’s real-time PCR and 89.9% compared to Francis’ real-time PCR. The agreement between the techniques was almost perfect according to the estimated Cohen’s kappa-index, even in symptomless almond trees. The developed immunological techniques represent sustainable and low-cost analysis tools, based on specific, homogeneous, and well-characterized MAbs, which can be obtained in unlimited quantities in a reproducible way and constitute a guarantee for the standardization of commercial kits. They are a valuable option within a polyphasic strategy for the detection of X. fastidiosa

Enrichment Double-Antibody Sandwich Indirect Enzyme-Linked Immunosorbent Assay That Uses a Specific Monoclonal Antibody for Sensitive Detection of Ralstonia solanacearum in Asymptomatic Potato Tubers

Sensitive and specific routine detection of Ralstonia solanacearum in symptomless potato tubers was achieved by efficient enrichment followed by a reliable double-antibody sandwich indirect enzyme-linked immunosorbent assay based on the specific monoclonal antibody 8B-IVIA. This monoclonal antibody reacted with 168 typical R. solanacearum strains and did not recognize 174 other pathogenic or unidentified bacteria isolated from potato. The optimized protocol included an initial enrichment step consisting of shaking the samples in modified Wilbrink broth for 72 h at 29°C. This step enabled specific detection by the enzyme-linked immunosorbent assay of 1 to 10 CFU of R. solanacearum per ml of initial potato extract. Analysis of 233 commercial potato lots by this method provided results that coincided with the results of conventional methods

Aggressive Citrus tristeza virus isolates in Chile are MCA13-positive and VT type, while mild isolates are MCA13-negative and T30 type

Citrus tristeza virus (CTV) was reported in the 1960's to affect Meyer lemon trees in Chile, but no field symptoms were observed. This study performed a complete biological, serological and molecular characterization of one hundred CTV isolates obtained from different hosts and geographical origins. Decline symptoms (DI) were not found on trees grafted onto sour orange. In Pica Oasis in northern Chile, stem pitting (SP) was found to affect grapefruit and Mexican lime trees. Most isolates present in the central area were considered mild (MCA-13-negative), while in the northern area aggressive isolates were observed and detected. Some of these isolates were capable of causing SP on grapefruit under field and greenhouse conditions and on sweet orange under greenhouse conditions. Almost all of these isolates were MCA13-positive and had nucleotide sequences associated with the VT genotype.El virus de la tristeza de los cítricos fue reportado el año 1960 afectando a árboles de limonero Meyer pero no se observaron síntomas a nivel de campo. Esta investigación tuvo como objetivo realizar una completa caracterización biológica, serológica y molecular de 100 aislados Chilenos de Citrus tristeza virus (CTV), previamente obtenidos desde diferentes hospederos y regiones geográficas. No se encontró síntomas de decaimiento (DI) en arboles injertados sobre naranjo amargo. En el Oasis de Pica, localidad de la zona norte de Chile, fue encontrada tristeza causando Stem pitting (SP) en árboles de pomelo y lima Mexicana. La mayoría de los aislados presentes en la zona central se consideraron leves (MCA-13 negativos), mientras que en la zona norte se observaron y se detectaron aislados agresivos, algunos capaces de causar SP en pomelo en condiciones campo e invernadero, y algunos capaces de causar SP en naranjo dulce bajo condiciones de invernadero, de los cuales casi todos fueron MCA-13 positivos y la secuencia de nucleótidos asociada al genotipo VT

The position of the major QTL for Citrus tristeza virus resistance is conserved among Citrus grandis, C. aurantium and Poncirus trifoliata

[EN] Citrus tristeza virus, CTV, is one of the most important citrus pathogens. Although CTV-resistant citrus rootstocks derived from Poncirus trifoliata are common, useful genetic resistance within the genus Citrus for scion improvement is very limited and no CTV-resistant sweet orange cultivar is yet available. Quantitative trait locus (QTL) analysis of the accumulation and distribution of CTV was carried out in a segregating population of 201 C. grandis x C. clementina hybrids derived from a reciprocal cross between two commercial varieties, Chandler (Ch) and Fortune (F), to genetically study the CTV resistance response. Chandler and 13 of its hybrids were found resistant to T-346 CTV isolate. The mortality of C. grandis x C. clementina hybrids, as well as CTV-challenged C. aurantium x P. trifoliata hybrids, was found to be related to the CTV resistance response. The type of cytoplasm (F or Ch) was not associated with the CTV resistance. A major QTL contributing around 24% of the total variance for CTV accumulation and spatial distribution was detected on linkage group 4b. Its position is conserved among C. grandis, C. aurantium and P. trifoliata. Seventy candidate genes of the CTV resistance response were obtained by transcriptomic bulk segregant analysis of resistant versus susceptible CTV-inoculated hybrids. Thirteen out of 28 of those candidates could be mapped on the C. grandis and/or C. clementina linkage maps. None was located on linkage group 4b, but four of them were found to be associated with CTV resistance through a minor QTL (CTVCh14) or epistatic interactions (CTVCh15, CTVCh17 and VIC).This work has been partially supported by grants RTA2006-0009-00-00, AGL2008-00197/AGR, Fondo Social Europeo (GPB) and IVIA (JFR). We thank Dr. J. Forment (IBMCP, UPV-CSIC) and J. Puchades (IVIA) for gene annotation and technical assistance, respectively.Asins, MJ.; Fernández Ribacoba, J.; Bernet, GP.; Gadea Vacas, J.; Cambra Alvarez, MJ.; Gorris, MT.; Carbonell, EA. (2012). The position of the major QTL for Citrus tristeza virus resistance is conserved among Citrus grandis, C. aurantium and Poncirus trifoliata. Molecular Breeding. 29(3):575-587. https://doi.org/10.1007/s11032-011-9574-xS575587293Aoki K, Kragler F, Xoconostle-Cazares B, Lucas WJ (2002) A subclass of 600 plant heat shock cognate 70 chaperones carries a motif that facilitates 601 trafficking through plasmodesmata. Proc Natl Acad Sci USA 99:16342–16347Asami T, Nakano T, Fujioka S (2005) Plant brassinosteroid hormones. Vitam Horm 72:479–504Asins MJ, Bernet GP, Ruiz C, Cambra M, Guerri J, Carbonell EA (2004) QTL analysis of Citrus Tristeza Virus-citradia interaction. Theor Appl Genet 108:603–611Asins MJ, Villalta I, Bernet GP, Carbonell EA (2009) Chapter 1. QTL analysis in plant breeding. 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Lateral flow immunoassay for on-site detection of <i>Xanthomonas arboricola</i> pv. <i>pruni</i> in symptomatic field samples

<div><p><i>Xanthomonas arboricola</i> pv. <i>pruni</i> is a quarantine pathogen and the causal agent of the bacterial spot disease of stone fruits and almond, a major threat to <i>Prunus</i> species. Rapid and specific detection methods are essential to improve disease management, and therefore a prototype of a lateral flow immunoassay (LFIA) was designed for the detection of <i>X</i>. <i>arboricola</i> pv. <i>pruni</i> in symptomatic field samples. It was developed by producing polyclonal antibodies which were then combined with carbon nanoparticles and assembled on nitrocellulose strips. The specificity of the LFIA was tested against 87 <i>X</i>. <i>arboricola</i> pv. <i>pruni</i> strains from different countries worldwide, 47 strains of other <i>Xanthomonas</i> species and 14 strains representing other bacterial genera. All <i>X</i>. <i>arboricola</i> pv. <i>pruni</i> strains were detected and cross-reactions were observed only with four strains of <i>X</i>. <i>arboricola</i> pv. <i>corylina</i>, a hazelnut pathogen that does not share habitat with <i>X</i>. <i>arboricola</i> pv. <i>pruni</i>. The sensitivity of the LFIA was assessed with suspensions from pure cultures of three <i>X</i>. <i>arboricola</i> pv. <i>pruni</i> strains and with spiked leaf extracts prepared from four hosts inoculated with this pathogen (almond, apricot, Japanese plum and peach). The limit of detection observed with both pure cultures and spiked samples was 10⁴ CFU ml^-1. To demonstrate the accuracy of the test, 205 samples naturally infected with <i>X</i>. <i>arboricola</i> pv. <i>pruni</i> and 113 samples collected from healthy plants of several different <i>Prunus</i> species were analyzed with the LFIA. Results were compared with those obtained by plate isolation and real time PCR and a high correlation was found among techniques. Therefore, we propose this LFIA as a screening tool that allows a rapid and reliable diagnosis of <i>X</i>. <i>arboricola</i> pv. <i>pruni</i> in symptomatic plants.</p></div