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Molecular Diagnosis and Diversity for Regulated Xanthomonas

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Molecular Methods in Diagnosis and Detection of Regulated Xanthomonads

This data is based on the review by Catara (2021), focused on the molecular methods for diagnosis, detection, and studies on the diversity of plant pathogenic Xanthomonas, concentrating especially on regulated pathogens in the European Union published as a collective effort of the Working Group 1 'Diagnostics & Diversity–Population Structure' from the EuroXanth COST Action CA16107.

Main protocols described for regulated xanthomonads based on amplification of specific target DNAs.

Disease Bacteria Conventional PCR qPCR1 IA2 Methods
Onion bacterial blight X. euvesicatoria pv. alli Robène-Soustrade (2010) Robène (2015) NA3
Citrus bacterial canker X. citri pv. citri
X. aurantifolii pathotypes B and C
Hartung (1996),
Cubero (2002),
Coletta-Filho (2006),
Suk Park (2006),
Kositcharoenkul (2011),
Fonseca (2019)
Mavrodieva (2004),
Cubero (2005),
Robène (2020)
Rigano (2010)
Bacterial spot of stone fruits, walnut blight, hazelnut blight X. arboricola pvs. pruni, corylina and juglandis Park (2010),
Fernandes (2017),
Jouen (2019),
Webber (2020)
Palacio-Bielsa (2011),
Garita-Cambronero (2017),
Martins (2019)
Bühlmann (2013),
Li (2021)
Bacterial leaf blight and bacterial leaf streak of rice X. oryzae pvs. oryzae and oryzicola Sakthivel (2001),
Kang (2008),
Lang (2010),
Cho (2011),
Shen (2012),
Singhal (2015),
Cui (2016)
Cho (2011),
Kang (2012)
Lang (2014)
Bacterial spot of tomato and sweet pepper X. euvesicatoria pvs. euvesicatoria and perforans,
X. hortorum pv. gardneri,
X. vesicatoria
Moretti (2009),
Araújo (2011),
Araújo (2013),
Beran (2013),
Koenraadt (2019),
Pečenka (2020)
Strayer (2016),
Pečenka (2020)
Larrea-Sarmiento (2018),
Strayer-Scherer (2019),
Stehlíková (2020)
Bacterial angular leaf spot of strawberry X. fragariae Hartung (1997),
Cruz (1999),
Stöger (2004),
Moltmann (2005)
Turechek (2008),
Vandroemme (2008),
Cubero (2009)
Wang (2016), Gétaz (2017),
Wang (2020),
Immanuel (2020)
Bacterial blight of anthurium and other aroids X. phaseoli pv. dieffenbachiae Khoodoo (2005),
Robène-Soustrade (2006),
Chabirand (2014)
Jouen (2019) Jun-Hai (2015)
Bacterial leaf spot of poinsettia X. axonopodis pv. poinsetticola Back (2015) NA NA
Bacterial leaf streak X. translucens pv. translucens Maes (1995) NA Langlois (2017)
Common blight of bean X. phaseoli pv. phaseoli,
X. citri pv. fuscans
Audy (1996) NA De Paiva (2020)

1 qPCR: quantitative real-time PCR; 2 IA: isothermal amplification: 3 NA: not applicable.


Araújo ER, Costa JR, Ferreira MASV, Quezado-Duval AM (2012). Simultaneous detection and identification of the Xanthomonas species complex associated with tomato bacterial spot using species-specific primers and multiplex PCR. J. Appl. Microbiol. 113: 1479-1490. DOI: 10.1111/j.1365-2672.2012.05431.x

Araújo ER, Ferreira MASV, Quezado-Duval AM (2013). Specific primers for Xanthomonas vesicatoria, a tomato bacterial spot causal agent. Eur. J. Plant Pathol. 137: 5-9. DOI: 10.1007/s10658-013-0225-4

Audy P, Braat CE, Saindon G, Huang HC, Laroche A (1996). A rapid and sensitive PCR-based assay for concurrent detection of bacteria causing common and halo blights in bean seed. Phytopathology 86: 361-366. DOI: 10.1094/Phyto-86-361

Back CG, Lee SY, Lee BJ, Yea MC, Kim SM, Kang IK, Cha JS, Jung HY (2015). Development of a species-specific PCR assay for three Xanthomonas species, causing bulb and flower diseases, based on their genome sequences. Plant Pathol. J. 31: 212-218. DOI: 10.5423/PPJ.OA.04.2015.0049

Beran P, Mráz I (2013). Species-specific PCR primers for detection of Xanthomonas vesicatoria. Crop. Prot. 43: 213-215. DOI: 10.1016/j.cropro.2012.08.008

Bühlmann A, Pothier JF, Tomlinson JA, Frey JE, Boonham N, Smits THM, Duffy B (2013). Genomics-informed design of loop-mediated isothermal amplification for detection of phytopathogenic Xanthomonas arboricola pv. pruni at the intraspecific level. Plant Pathol. 62: 475-484. DOI: 10.1111/j.1365-3059.2012.02654.x

Chabirand A, Jouen E, Pruvost O, Chiroleu F, Hostachy B, Bergsma-Vlami M, Bianchi G, Cozzolino L, Elphinstone J, Holeva M, Manole F, Martini P, Matoušková H, Minatchy J, Op de Beeck G, Poliakoff F, Sigillo L, Siverio F, Van Vaerenbergh J, Laurentie M, Robène-Soustrade I (2014). Comparative and collaborative studies for the validation of a nested PCR for the detection of Xanthomonas axonopodis pv. dieffenbachiae from Anthurium samples. Plant Pathol. 63: 20-30. DOI: 10.1111/ppa.12083

Cho MS, Kang MJ, Kim CK, Seol YJ, Hahn JH, Park SC, Hwang DJ, Ahn TY, Park DH, Lim CK, Park DS (2011). Sensitive and specific detection of Xanthomonas oryzae pv. oryzae by real-time bio-PCR using pathovar-specific primers based on an rhs family gene. Plant Dis. 95: 589-594. DOI: 10.1094/PDIS-06-10-0399

Coletta-Filho HD, Takita MA, De Souza AA, Neto JR, Destéfano SAL, Hartung JS, Machado MA (2006). Primers based on the rpf gene region provide improved detection of Xanthomonas axonopodis pv. citri in naturally and artificially infected citrus plants. J. Appl. Microbiol. 100: 279-285. DOI: 10.1111/j.1365-2672.2005.02787.x

Cruz L, Sousa-Santos M (1999). Optimizing PCR technique for large scale diagnosis of angular leaf spot of strawberry in Fragaria plants. Petria 9: 77-80.

Cubero J, Ayllón MA, Gell I, Melgarejo P, De Cal A, Martín-Sánchez PM, Pérez-Jiménez RM, Soria C, Segundo E, Larena I (2009). Detection of strawberry pathogens by real-time PCR. Acta Hortic. 842: 263-266. DOI: 10.17660/ActaHortic.2009.842.44

Cubero J, Graham JH (2002). Genetic relationship among worldwide strains of Xanthomonas causing canker in citrus species and design of new primers for their identification by PCR. Appl. Environ. Microbiol. 68: 1257-1264. DOI: 10.1128/AEM.68.3.1257-1264.2002

Cubero J, Graham JH (2005). Quantitative real-time polymerase chain reaction for bacterial enumeration and allelic discrimination to differentiate Xanthomonas strains on citrus. Phytopathology 95: 1333-1340. DOI: 10.1094/PHYTO-95-1333

Cui Z, Ojaghian MR, Tao Z, Kakar KU, Zeng J, Zhao W, Duan Y, Vera Cruz CM, Li B, Zhu B, Xie G (2016). Multiplex PCR assay for simultaneous detection of six major bacterial pathogens of rice. J. Appl. Microbiol.120,1357–1367.DOI: 10.1111/jam.13094

De Paiva BAR, Wendland A, Teixeira NC, Ferreira MASV (2020). Rapid detection of Xanthomonas citripv. fuscans and Xanthomonas phaseoli pv. phaseoli in common bean by Loop-Mediated Isothermal Amplification. Plant Dis. 104: 198-203. DOI: 10.1094/PDIS-02-19-0325-RE

Fernandes C, Albuquerque P, Sousa R, Cruz L, Tavares F (2017). Multiple DNA markers for identification of Xanthomonas arboricola pv. juglandis isolates and its direct detection in plant samples. Plant Dis. 101: 858-865. DOI: 10.1094/PDIS-10-16-1481-RE

Fonseca, NP, Felestrino ÉB, Caneschi WL, Sanchez AB, CordeiroIF, Lemes CGC, Assis RAB, Carvalho FMS, Ferro JA, Varani AM, Belasque J, Setubal JC, Telles GP, Aguena DS, Almeida​ NF, Moreira​ LM (2019). Detection and identification of Xanthomonas pathotypes associated with citrus diseases using comparative genomics and multiplex PCR. PeerJ 7: e7676. DOI: 10.7717/peerj.7676

Garita-Cambronero J, Palacio-Bielsa A, López MM, Cubero J (2017). Pan-genomic analysis permits differentiation of virulent and non-virulent strains of Xanthomonas arboricola that cohabit Prunus spp. and elucidate bacterial virulence factors. Front. Microbiol. 8: 573. DOI: 10.3389/fmicb.2017.00573

Gétaz M, Bühlmann A, Schneeberger PHH, Van Malderghem C, Duffy B, Maes M, Pothier JF, Cottyn B (2017). A diagnostic tool for improved detection of Xanthomonas fragariae using a rapid and highly specific LAMP assay designed with comparative genomics. Plant Pathol. 66: 1094-1102. DOI: 10.1111/ppa.12665

Hartung J, Pruvost OP, Villemot I, Alvarez A (1996). Rapid and sensitive colorimetric detection of Xanthomonas axonopodis pv. citri by immunocapture and a nested polymerase chain reaction assay. Phytopathology 86: 95. DOI: 10.1094/Phyto-86-95

Hartung JS, Pooler MR (1997). Immunocapture and multiplexed-PCR assay for Xanthomonas fragariae, causal agent of angular leafspot disease. Int. Soc. Hortic. Sci. 439: 821-828. DOI: 10.17660/ActaHortic.1997.439.136

Immanuel T, Taylor R, Keeling S, Brosnahan C, Alexander B (2020). Discrimination between viable and dead Xanthomonas fragariae in strawberry using viability PCR. J. Phytopathol. 168: 363-373. DOI: 10.1111/jph.12900

Jouen E, Chiroleu F, Maillot-Lebon V, Chabirand A, Merion S, Boyer C, Pruvost O, Robène I (2019). A duplex quantitative real-time PCR assay for the detection and quantification of Xanthomonas phaseoli pv. dieffenbachiae from diseased and latently infected anthurium tissue. J. Microbiol. Methods 161: 74-83. DOI: j.mimet.2019.03.003

Jun-Hai N, Yue-Rong G, Jun-Mei Y, Qing-Yun L, Guang-Sui Y, Cun W, Yu R (2015). Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of bacterial blight pathogen (Xanthomonas axonopodis pv. dieffenbachiae) in anthurium. Eur. J. Plant Pathol. 142: 801-813. DOI: 10.1007/s10658-015-0653-4

Kang MJ, Kim MH, Hwang DJ, Cho MS, Seol Y, Hahn JH, Ryu S, Park DS (2012). Quantitative in planta PCR assay for specific detection of Xanthomonas oryzae pv. oryzicola using putative membrane protein based primer set. Crop. Prot. 40: 22-27. DOI: 10.1016/j.cropro.2012.04.014

Kang MJ, Shim JK, Cho MS, Seol YJ, Hahn JH, Hwang DJ, Park DS (2008). Specific detection of Xanthomonas oryzae pv. oryzicola in infected rice plant by use of PCR assay targeting a membrane fusion protein gene. J. Microbiol. Biotechnol. 18: 1492-1495. PMID: 18852502

Khoodoo MHR, Sahin F, Jaufeerally-Fakim Y (2005). Sensitive detection of Xanthomonas axonopodis pv. dieffenbachiae on Anthurium andreanum by immunocapture-PCR (IC-PCR) using primers designed from sequence characterized amplified regions (SCAR) of the blight pathogen. Eur. J. Plant Pathol. 112: 379-390. DOI: 10.1007/s10658-005-7062-z

Koenraadt H, van Betteray B, Germain R, Hiddink G, Jones JB, Oosterhof J (2009). Development of specific primers for the molecular detection of bacterial spot of pepper and tomato. Acta Hortic. 808: 99-102. DOI: 10.17660/ActaHortic.2009.808.13

Kositcharoenkul N, Chatchawankanphanich O, Bhunchoth A, Kositratana W (2011). Detection of Xanthomonas citri subsp. citri from field samples using single-tube nested PCR. Plant Pathol. 60: 436-442. DOI: 10.1111/j.1365-3059.2010.02390.x

Lang JM, Hamilton JP, Diaz MGQ, Van Sluys MA, Burgos MRG, Vera Cruz CM, Buell CR, Tisserat NA, Leach JE (2010). Genomics-based diagnostic marker development for Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola. Plant Dis. 94: 311-319. DOI: 10.1094/PDIS-94-3-0311

Lang JM, Langlois P, Nguyen MHR, Triplett LR, Purdie L, Holton TA, Djikeng A, Vera Cruz CM, Verdier V, Leach JE (2014). Sensitive detection of Xanthomonas oryzae pathovars oryzae and oryzicolaby loop-mediated isothermal amplification. Appl. Environ. Microbiol. 80: 4519-4530. DOI: 10.1128/AEM.00274-14

Langlois PA, Snelling J, Hamilton JP, Bragard C, Koebnik R, Verdier V, Triplett LR, Blom J, Tisserat NA, Leach JE (2017). Characterization of the Xanthomonas translucens complex using draft genomes, comparative genomics, phylogenetic analysis, and diagnostic LAMP assays. Phytopathology 107: 519-527. DOI: 10.1094/PHYTO-08-16-0286-R

Larrea-Sarmiento A, Dhakal U, Boluk G, Fatdal L, Alvarez A, Strayer-Scherer A, Paret M, Jones J, Jenkins D, Arif M (2018). Development of a genome-informed loop-mediated isothermal amplification assay for rapid and specific detection of Xanthomonas euvesicatoria. Sci. Rep. 8: 14298. DOI: 10.1038/s41598-018-32295-4

Li W, Lee SY, Back CG, Ten LN, Jung HY (2019). Loop-mediated isothermal amplification for the detection of Xanthomonas arboricola pv. pruni in peaches. Plant Pathol. J. 35: 635-643. DOI: 10.5423/PPJ.OA.07.2019.0197

Maes M, Garbeva P (1995). Development of a PCR-based detection method for Xanthomonas campestris pv. translucens. EPPO Bull. 25: 203-209. DOI: 10.1111/j.1365-2338.1995.tb01459.x

Martins L, Fernandes C, Albuquerque P, Tavares F (2019). Assessment of Xanthomonas arboricola pv. juglandis bacterial load in infected walnut fruits by quantitative PCR. Plant Dis. 103: 2577-2586. DOI: 10.1094/PDIS-12-18-2253-RE

Mavrodieva V, Levy L, Gabriel DW (2004). Improved sampling methods for real-time polymerase chain reaction diagnosis of citrus canker from field samples. Phytopathology 94: 61-68. DOI: 10.1094/PHYTO.2004.94.1.61

Moltmann E, Zimmermann C (2005). Detection of Xanthomonas fragariae in symptomless strawberry plants by nested PCR. EPPO Bull. 35: 53-54. DOI: 10.1111/j.1365-2338.2005.00812.x

Moretti C, Amatulli MT, Buonaurio R (2009). PCR-based assay for the detection of Xanthomonas euvesicatoria causing pepper and tomato bacterial spot. Lett. Appl. Microbiol. 49: 466-471. DOI: 10.1111/j.1472-765X.2009.02690.x

Palacio-Bielsa A, Cubero J, Cambra MA, Collados R, Berruete IM, López MM (2011). Development of an efficient real-time quantitative PCR protocol for detection of Xanthomonas arboricola pv. pruni in Prunus species. Appl. Environ. Microbiol. 77: 89-97. DOI: 10.1128/AEM.01593-10

Park SY, Lee YS, Koh YJ, Hur JS, Jung JS (2010). Detection of Xanthomonas arboricola pv. pruni by PCR using primers based on DNA sequences related to the hrp genes. J. Microbiol. 48: 554-558. DOI: 10.1007/s12275-010-0072-3

Pečenka J, Kocanová M, Baránek M, Gazdík F, Ragasová L, Peňázová E, Ĉechová J, Beran P, Eichmeier A (2020). Species-specific PCR primers for the detection of poorly distinguishable Xanthomonas euvesicatoria. Crop. Prot. 127: 104978. DOI: 10.1016/j.cropro.2019.104978

Rigano LA, Marano MR, Castagnaro AP, Do Amaral AM, Vojnov AA (2010). Rapid and sensitive detection of Citrus Bacterial Canker by loop-mediated isothermal amplification combined with simple visual evaluation methods. BMC Microbiol. 10: 176. DOI: 10.1186/1471-2180-10-176

Robène I, Perret M, Jouen E, Escalon A, Maillot MV, Chabirand A, Moreau A, Laurent A, Chiroleu F, Pruvost O (2015). Development and validation of a real-time quantitative PCR assay to detect Xanthomonas axonopodis pv. allii from onion seed. J. Microbiol. Methods. 114: 78-86. DOI: 10.1016/j.mimet.2015.04.017

Robène I, Maillot-Lebon V, Chabirand A, Moreau A, Becker N, Moumène A, Rieux A, Campos P, Gagnevin L, Gaudeul M, Baider C, Chiroleu F, Pruvost O (2020). Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants. BMC Microbiol. 20: 296. DOI: 10.1186/s12866-020-01972-8

Robène-Soustrade I, Laurent P, Gagnevin L, Jouen E, Pruvost O (2006). Specific detection of Xanthomonas axonopodis pv. dieffenbachiae in anthurium (Anthurium andreanum) tissues by nested PCR. Appl. Environ. Microbiol. 72: 1072-1078. DOI: 10.1128/AEM.72.2.1072-1078.2006

Robène-Soustrade I, Legrand D, Gagnevin L, Chiroleu F, Laurent A, Pruvost O (2020). Multiplex nested PCR for detection of Xanthomonas axonopodis pv. allii from onion seeds. Appl. Environ. Microbiol. 76: 2697-2703. DOI: 10.1128/AEM.02697-09

Sakthivel N, Mortensen CN, Mathur SB (2001). Detection of Xanthomonas oryzae pv. oryzae in artificially inoculated and naturally infected rice seeds and plants by molecular techniques. Appl. Microbiol. Biotechnol. 56: 435-441. DOI: 10.1007/s002530100641

Shen YP, Zou LF, Li YR, Zou HS, Liu XL, Chen GY (2012). Xoryp_08180 of Xanthomonas oryzae pv. oryzicola, encoding a hypothetical protein, is regulated by HrpG and HrpX and required for full virulence in rice. J. Integr. Agric. 11: 600-610. DOI: 10.1016/S2095-3119

Singhal N, Kumar M, Kanaujia PK, Virdi JS (2015). MALDI-TOF mass spectrometry: An emerging technology for microbial identification and diagnosis. Front. Microbiol. 6: 791. DOI: 10.3389/fmicb.2015.00791

Stehlíková D, Beran P, Cohen SP, Curn V (2020). Development of real-time and colorimetric loop mediated isothermal amplification assay for detection of Xanthomonas gardneri. Microorganisms 8: 1301. DOI: 10.3390/microorganisms8091301

Stöger A, Ruppitsch W (2004). A rapid and sensitive method for the detection of Xanthomonas fragariae, causal agent of angular leafspot disease in strawberry plants. J. Microbiol. Methods 58: 281-284. DOI: 10.1016/j.mimet.2004.04.002

Strayer AL, Jeyaprakash A, Minsavage GV, Timilsina S, Vallad GE, Jones JB, Paret ML (2016). A multiplex real-time PCR assay differentiates four Xanthomonas species associated with bacterial Spot of tomato. Plant Dis. 100: 1660-1668. DOI: 10.1094/PDIS-09-15-1085-RE

Strayer-Scherer A, Jones JB, Paret ML (2019). Recombinase polymerase amplification assay for field detection of tomato bacterial spot pathogens. Phytopathology 109: 690-700. DOI: 10.1094/PHYTO-03-18-0101-R

Suk Park D, Wook Hyun J, Jin Park Y, Sun Kim J, Wan Kang H, Ho Hahn J, Joo Go S (2006). Sensitive and specific detection of Xanthomonas axonopodis pv. citri by PCR using pathovar specific primers based on hrpW gene sequences. Microbiol. Res. 161: 145-149. DOI: 10.1016/j.micres.2005.07.005

Turechek WW, Hartung JS, McCallister J (2008). Development and optimization of a real-time detection assay for Xanthomonas fragariae in strawberry crown tissue with receiver operating characteristic curve analysis. Phytopathology 98: 359-368. DOI: 10.1094/PHYTO-98-3-0359

Vandroemme J, Baeyen S, Van Vaerenbergh J, De Vos, Maes M (2008). Sensitive real-time PCR detection of Xanthomonas fragariae in strawberry plants. Plant Pathol. 57: 438-444. DOI: 10.1111/j.1365-3059.2007.01813.x

Wang H, Turechek WW (2016). A loop-mediated isothermal amplification assay and sample preparation procedure for sensitive detection of Xanthomonas fragariae in strawberry. PLoS ONE 11: e0147122. DOI: 10.1371/journal.pone.0147122

Wang H, Turechek WW (2020). Detection of viable Xanthomonas fragariae cells in strawberry using propidium monoazide and long-amplicon quantitative PCR. Plant Dis. 104: 1105-1112. DOI: 10.1094/PDIS-10-19-2248-RE

Webber JB, Putnam M, Serdani M, Pscheidt JW, Wiman NG, Stockwell VO (2020). Characterization of isolates of Xanthomonas arboricola pv. corylina, the causal agent of bacterial blight, from Oregon hazelnut orchards. J. Plant Pathol. 102: 799-812. DOI: 10.1007/s42161-020-00505-6

Further reading

Catara V, Cubero J, Pothier JF, Bosis E, Bragard C, Đermić E, Holeva MC, Jacques MA, Petter F, Pruvost O, Robène I, Studholme DJ, Tavares F, Vicente JG, Koebnik R, Costa J (2021) Trends in molecular diagnosis and diversity studies for phytosanitary regulated Xanthomonas. Microorganisms 9: 862. DOI: 10.3390/microorganisms9040862

diagnostics/dna_protocols.txt · Last modified: 2022/05/05 09:40 by rkoebnik