Real-time PCR applied to study on plant pathogens: potential applications in diagnosis – a review S.M., Loit E., Mänd M., Mansouripour S.M. (2015): Real-time PCR applied to study on plant pathogens: potential applications in diagnosis – a review. Plant Protect. Sci., 51: 177-190.
download PDF
Quantitative real-time PCR (qPCR) technique incorporates traditional polymerase chain reaction (PCR) efficiency with the production of a specific fluorescent signal, measuring the kinetics of the reaction in the early PCR phases and providing quantification of specific targets in various environmental samples. There are an increasing number of chemistries to detect PCR products, which are widely used in plant pathology as they cluster into the amplicon sequence non-specific and sequence-specific techniques. In this review, we illustrate a general description of major chemistries and discuss some considerations for assay development as it applies for a wide range of applications in epidemiological studies. The technique has become the gold standard for early detection of pathogens and a fundamental tool in the research laboratory.
Fernández Acero Francisco Javier, Carbú María, El-Akhal Mohamed Rabie, Garrido Carlos, González-Rodríguez Victoria E., Cantoral Jesús M. (2011): Development of Proteomics-Based Fungicides: New Strategies for Environmentally Friendly Control of Fungal Plant Diseases. International Journal of Molecular Sciences, 12, 795-816
Arya Manit, Shergill Iqbal S, Williamson Magali, Gommersall Lyndon, Arya Neehar, Patel Hitendra RH (2005): Basic principles of real-time quantitative PCR. Expert Review of Molecular Diagnostics, 5, 209-219
Baric Sanja (2012): Quantitative Real-Time PCR Analysis of ‘Candidatus Phytoplasma mali’ Without External Standard Curves. Erwerbs-Obstbau, 54, 147-153
Barnes C. W., Szabo L. J. (2007): Detection and Identification of Four Common Rust Pathogens of Cereals and Grasses Using Real-Time Polymerase Chain Reaction. Phytopathology, 97, 717-727
Bilodeau G. J., Lévesque C. A., de Cock A. W. A. M., Duchaine C., Brière S., Uribe P., Martin F. N., Hamelin R. C. (2007): Molecular Detection of Phytophthora ramorum by Real-Time Polymerase Chain Reaction Using TaqMan, SYBR Green, and Molecular Beacons. Phytopathology, 97, 632-642
Bilodeau Guillaume J., Koike Steven T., Uribe Pedro, Martin Frank N. (2012): Development of an Assay for Rapid Detection and Quantification of Verticillium dahliae in Soil. Phytopathology, 102, 331-343
Boa-Sorte P.M.F., Simoes-Araujo J.L., de Melo L.H.V., de Souza Galisa P., Leal L., Baldani J.I., Baldani V.L.D. (2014): Development of a real-time PCR assay for the detection and quantification of Gluconacetobacter diazotrophicus in sugarcane grown under field conditions. African Journal of Microbiology Research, 8(31): 2937–2946.
Boben Jana, Kramberger Petra, Petrovič Nataša, Cankar Katarina, Peterka Matjaž, Štrancar Aleš, Ravnikar Maja (2007): Detection and quantification of Tomato mosaic virus in irrigation waters. European Journal of Plant Pathology, 118, 59-71
Bonants Peter J.M., van Gent-Pelzer Marga P.E., Hooftman Rien, Cooke David E.L., Guy Dave C., Duncan Jim M. (2004): A Combination of Baiting and Different PCR Formats, Including Measurement of Real-Time Quantitative Fluorescence, For the Detection of Phytophthora fragariae in Strawberry Plants. European Journal of Plant Pathology, 110, 689-702
Boonham N, Pérez L.González, Mendez M.S, Peralta E.Lilia, Blockley A, Walsh K, Barker I, Mumford R.A (2004): Development of a real-time RT-PCR assay for the detection of Potato spindle tuber viroid. Journal of Virological Methods, 116, 139-146
Capote N., Pastrana A.M., Aguado A., Torres P.S. (2012): Molecular tools for detection of plant pathogenic fungi and fungicide resistance. In: Cumagun C.J. (ed.): Agricultural and Biological Sciences “Plant Pathology”. Rijeka, InTech: 151–202.
Cooke D.E.L., Schena L., Cacciola S.O. (2007): Tools to detect, identify and monitor Phytophthora species in natural ecosystems. Journal of Plant Pathology, 89: 13–28.
CHEN Wei, DAI Jin, ZHANG Huawei, JIAO Honghong, CHENG Julong, WU Yunfeng (2014): Concentration and detection of tobacco etch virus from irrigation water using real-time PCR. TURKISH JOURNAL OF AGRICULTURE AND FORESTRY, 38, 471-477
Seok Cho Min, Jeon Yong Ho, Jung Kang Man, Ahn Hong Il, Baek Hyung-Jin, Wang Na Young, Mi Choi Yu, San Kim Tae, Suk Park Dong (2010): Sensitive and specific detection of phaseolotoxigenic and nontoxigenic strains of Pseudomonas syringae pv. phaseolicola by TaqMan real-time PCR using site-specific recombinase gene sequences. Microbiological Research, 165, 565-572
Clément J.A.J., Baldwin T.K., Magalon H., Glais I., Gracianne C., Andrivon D., Jacquot E. (2013): Specific detection and quantification of virulent/avirulent Phytophthora infestans isolates using a real-time PCR assay that targets polymorphisms of the Avr3a gene. Letters in Applied Microbiology, 56, 322-332
Cullen D.W., Hirsch P.R. (1998): Simple and rapid method fordirect extraction of microbial DNA fromsoil for PCR. Soil Biology and Biochemistry, 30, 983-993
Dai J., Peng H., Chen W., Cheng J., Wu Y. (2013): Development of multiplex real-time PCR for simultaneous detection of three Potyviruses in tobacco plants. Journal of Applied Microbiology, 114, 502-508
Didenko V.V. (2001): DNA probes using fluorescence resonance energy transfer (FRET): designs and applications. BioTechniques, 31: 1106–1121.
Diguta Camélia Filofteia, Rousseaux Sandrine, Weidmann Stéphanie, Bretin Nicolas, Vincent Béatrice, Guilloux-Benatier Michèle, Alexandre Hervé (2010): Development of  a qPCR assay for specific quantification of Botrytis cinerea on grapes. FEMS Microbiology Letters, 313, 81-87
Dreo Tanja, Pirc Manca, Ravnikar Maja (2012): Real-time PCR, a method fit for detection and quantification of Erwinia amylovora. Trees, 26, 165-178
Espy M. J., Uhl J. R., Sloan L. M., Buckwalter S. P., Jones M. F., Vetter E. A., Yao J. D. C., Wengenack N. L., Rosenblatt J. E., Cockerill F. R., Smith T. F. (): Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing. Clinical Microbiology Reviews, 19, 165-256
Finetti-Sialer M. M., Ciancio A. (2005): Isolate-Specific Detection of Grapevine fanleaf virus from Xiphinema index Through DNA-Based Molecular Probes. Phytopathology, 95, 262-268
Francis Marta, Lin Hong, Rosa Juan Cabrera-La, Doddapaneni Harshavardhan, Civerolo Edwin L. (2006): Genome-based PCR Primers for Specific and Sensitive Detection and Quantification of Xylella fastidiosa. European Journal of Plant Pathology, 115, 203-213
Fredslund Jakob, Lange Mette (): Primique: automatic design of specific PCR primers for each sequence in a family. BMC Bioinformatics, 8, 369-
Galetto L., Bosco D., Marzachi C. (2005): Universal and group-specific real-time PCR diagnosis of flavescence doree (16Sr-V), bois noir (16Sr-XII) and apple proliferation (16Sr-X) phytoplasmas from field-collected plant hosts and insect vectors. Annals of Applied Biology, 147, 191-201
Garrido C., Carbú M., Fernández-Acero F. J., Boonham N., Colyer A., Cantoral J. M., Budge G. (2009): Development of protocols for detection of Colletotrichum acutatum and monitoring of strawberry anthracnose using real-time PCR. Plant Pathology, 58, 43-51
Garrido C., Acero F.G.F., Carbu M., Rodriguez V.E.G., Liniero E., Cantoral J.M. (2012): Molecular microbiology applied to the study of phytopathogenic fungi. In: Magdeldin S. (ed.): Biochemistry, Genetics and Molecular Biology. Rijeka, InTech: 139–156.
Giulietti Annapaula, Overbergh Lut, Valckx Dirk, Decallonne Brigitte, Bouillon Roger, Mathieu Chantal (2001): An Overview of Real-Time Quantitative PCR: Applications to Quantify Cytokine Gene Expression. Methods, 25, 386-401
Goud J.C., Termorshuizen A.J. (2003): Quality of methods to quantify microsclerotia of Verticillium dahliae in soil. European Journal of Plant Pathology, 109: 523– 534.
Hadidi A., Levy L., Podleckis E.V. (1995): Polymerase chain reaction technology in plant pathology. In: Singh R.P., Singh U.S. (eds): Molecular Methods in Plant Pathology. London, CRC Press Inc.: 167–187.
Haegi Anita, Catalano Valentina, Luongo Laura, Vitale Salvatore, Scotton Michele, Ficcadenti Nadia, Belisario Alessandra (2013): A Newly Developed Real-Time PCR Assay for Detection and Quantification of Fusarium oxysporum and Its Use in Compatible and Incompatible Interactions with Grafted Melon Genotypes. Phytopathology, 103, 802-810
Harper S. J., Ward L. I., Clover G. R. G. (2010): Development of LAMP and Real-Time PCR Methods for the Rapid Detection of Xylella fastidiosa for Quarantine and Field Applications. Phytopathology, 100, 1282-1288
Hussain Touseef, Singh Bir Pal, Anwar Firoz (2014): A quantitative Real Time PCR based method for the detection of Phytophthora infestans causing Late blight of potato, in infested soil. Saudi Journal of Biological Sciences, 21, 380-386
Hodgetts J., Boonham N., Mumford R., Dickinson M. (): Panel of 23S rRNA Gene-Based Real-Time PCR Assays for Improved Universal and Group-Specific Detection of Phytoplasmas. Applied and Environmental Microbiology, 75, 2945-2950
Hyndman D.L., Mitsuhashi M. (2003): PCR primer design. In: Bartlett M.S., Stirling D. (eds): PCR Protocols. Series: Methods in Molecular Biology, Vol. 226. 2nd Ed. New York, Humana Press: 81–88.
Ingle C. A., Kushner S. R. (1996): Development of an in vitro mRNA decay system for Escherichia coli: Poly(A) polymerase I is necessary to trigger degradation. Proceedings of the National Academy of Sciences, 93, 12926-12931
Ippolito Antonio, Schena Leonardo, Nigro Franco, Soleti ligorio Vincenza, Yaseen Thaer (2004): Real-time detection of Phytophthora nicotianae and P. citrophthorain citrus roots and soil. European Journal of Plant Pathology, 110, 833-843
Ishiguro T., Saitoh J., Yawata H., Yamagishi H., Iwasaki S., Mitoma Y. (1995): Homogeneous Quantitative Assay of Hepatitis C Virus RNA by Polymerase Chain Reaction in the Presence of a Fluorescent Intercalater. Analytical Biochemistry, 229, 207-213
Johnson Kameka L., Walcott Ron R. (2012): Progress Towards a Real-time PCR Assay for the Simultaneous Detection of Clavibacter michiganensis subsp. michiganensis and Pepino mosaic virus in Tomato Seed. Journal of Phytopathology, 160, 353-363
Kałużna Monika, Puławska Joanna, Mikiciński Artur (2013): Evaluation of methods for erwinia amylovora detection. Journal of Horticultural Research, 21, -
Lamarche Josyanne, Stewart Don, Pelletier Gervais, Hamelin Richard C., Tanguay Philippe (2014): Real-time PCR detection and discrimination of the Ceratocystis coerulescens complex and of the fungal species from the Ceratocystis polonica complex validated on pure cultures and bark beetle vectors. Canadian Journal of Forest Research, 44, 1103-1111
Li Mingzhu, Ishiguro Yasushi, Otsubo Kayoko, Suzuki Hirofumi, Tsuji Tomoko, Miyake Noriyuki, Nagai Hirofumi, Suga Haruhisa, Kageyama Koji (2014): Monitoring by real-time PCR of three water-borne zoosporic Pythium species in potted flower and tomato greenhouses under hydroponic culture systems. European Journal of Plant Pathology, 140, 229-242
Lievens B., Grauwet T.J.M.A., Cammue B.P.A., Thomma B.P.H.J. (2005): Recent developments in diagnostics of plant pathogens: a review. Recent Research Developments in Microbiology, 9: 57–79.
López-Fabuel Irene, Wetzel Thierry, Bertolini Edson, Bassler Alexandra, Vidal Eduardo, Torres Luis B., Yuste Alberto, Olmos Antonio (2013): Real-time multiplex RT-PCR for the simultaneous detection of the five main grapevine viruses. Journal of Virological Methods, 188, 21-24
Luigi Marta, Faggioli Francesco (2011): Development of quantitative real-time RT-PCR for the detection and quantification of Peach latent mosaic viroid. European Journal of Plant Pathology, 130, 109-116
Mahuku George S., Platt H. W. (2002): QuantifyingVerticillium dahliae in soils collected from potato fields using a competitive PCR assay. American Journal of Potato Research, 79, 107-117
Martin Robert R., James Delano, Lévesque C. André (2000): I MPACTS OF M OLECULAR D IAGNOSTIC T ECHNOLOGIES ON P LANT D ISEASE M ANAGEMENT. Annual Review of Phytopathology, 38, 207-239
Martin Frank N., Tooley Paul W., Blomquist Cheryl (2004): Molecular Detection of Phytophthora ramorum , the Causal Agent of Sudden Oak Death in California, and Two Additional Species Commonly Recovered from Diseased Plant Material. Phytopathology, 94, 621-631
Mavrodieva Vessela, Levy Laurene, Gabriel Dean W. (2004): Improved Sampling Methods for Real-Time Polymerase Chain Reaction Diagnosis of Citrus Canker from Field Samples. Phytopathology, 94, 61-68
Mbofung G. C. Y., Fessehaie A., Bhattacharyya M. K., Leandro L. F. S. (2011): A New TaqMan Real-Time Polymerase Chain Reaction Assay for Quantification of Fusarium virguliforme in Soil. Plant Disease, 95, 1420-1426
McCartney H Alastair, Foster Simon J, Fraaije Bart A, Ward Elaine (2003): Molecular diagnostics for fungal plant pathogens. Pest Management Science, 59, 129-142
Mehle N., Nikolic P., Gruden K., Ravnikar M., Dermastia M. (2012): Real-time PCR for specific detection of three Phytoplasmas from the apple proliferation group. In: Dickinson M., Hodgetts J. (eds): Phytoplasma: Methods and Protocols. Series: Methods in Molecular Biology, Vol. 938. New York, Humana Press: 269–281.
Mhlanga Musa M., Malmberg Lovisa (2001): Using Molecular Beacons to Detect Single-Nucleotide Polymorphisms with Real-Time PCR. Methods, 25, 463-471
Miller Daniel N. (2001): Evaluation of gel filtration resins for the removal of PCR-inhibitory substances from soils and sediments. Journal of Microbiological Methods, 44, 49-58
Montes-Borrego Miguel, Muñoz-Ledesma Francisco J., Jiménez-Díaz Rafael M., Landa Blanca B. (2011): Real-Time PCR Quantification of Peronospora arborescens , the Opium Poppy Downy Mildew Pathogen, in Seed Stocks and Symptomless Infected Plants. Plant Disease, 95, 143-152
Morrison T.B., Weis J.J., Wittwer C.T. (1998): Quantification of low-copy transcripts by continuous SYBR green I monitoring during amplification. BioTechniques, 24: 954–962.
Narayanasamy P. (2011): Detection of Virus and Viroid Pathogens in Plants. In: Microbial Plant Pathogens-Detection and Disease Diagnosis. Viral and Viroid Pathogens, Vol. 3. Dordrecht, Springer: 7–220.
Okubara Patricia A., Schroeder Kurtis L., Paulitz Timothy C. (2005): Real-time polymerase chain reaction: applications to studies on soilborne pathogens. Canadian Journal of Plant Pathology, 27, 300-313
Olexová Lídia, Kuchta Tomš?, Dovicovicová Lubomira (2004): Comparison of three types of methods for the isolation of DNA from flours, biscuits and instant paps. European Food Research and Technology, 218, 390-393
Palacio-Bielsa A., Cubero J., Cambra M. A., Collados R., Berruete I. M., Lopez M. M. (): Development of an Efficient Real-Time Quantitative PCR Protocol for Detection of Xanthomonas arboricola pv. pruni in Prunus Species. Applied and Environmental Microbiology, 77, 89-97
Papayiannis Lambros C. (2014): Diagnostic real-time RT-PCR for the simultaneous detection of Citrus exocortis viroid and Hop stunt viroid. Journal of Virological Methods, 196, 93-99
Parisi Olivier, Lepoivre Philippe, Jijakli M. Haissam (2011): Development of a Quick Quantitative Real-Time PCR for the In Vivo Detection and Quantification of Peach latent mosaic viroid. Plant Disease, 95, 137-142
Parker M. L., McDonald M. R., Boland G. J. (2014): Evaluation of Air Sampling and Detection Methods to Quantify Airborne Ascospores of Sclerotinia sclerotiorum. Plant Disease, 98, 32-42
Pelosi Carla S., Lourenço Mirian V., Silva Melissa, Santos Alexandre Z., França Suzelei C., Marins Mozart (2013): Development of a Taqman real-time PCR assay for detection of Leifsonia xyli subsp xyli. Tropical Plant Pathology, 38, 343-345
Reeleder R.D., Capell B.B., Tomlinson L.D., Hickey W.J. (2003): The extraction of fungal DNA from multiple large soil samples. Canadian Journal of Plant Pathology, 25, 182-191
Rizza S., Nobile G., Tessitori M., Catara A., Conte E. (2009): Real time RT-PCR assay for quantitative detection of Citrus viroid III in plant tissues. Plant Pathology, 58, 181-185
Roberts Cassie A, Dietzgen Ralf G, Heelan Lisa A, Maclean Donald J (2000): Real-time RT-PCR fluorescent detection of tomato spotted wilt virus. Journal of Virological Methods, 88, 1-8
Schaad Norman W., Frederick Reid. D., Shaw Joe, Schneider William L., Hickson Robert, Petrillo Michael D., Luster Douglas G. (2003): A DVANCES IN M OLECULAR -B ASED D IAGNOSTICS IN M EETING C ROP B IOSECURITY AND P HYTOSANITARY I SSUES *. Annual Review of Phytopathology, 41, 305-324
Schena L., Ippolito A. (2003): Rapid and sensitive detection of Rosellinia necatrix in roots and soils by real time Scorpion-PCR. Journal of Plant Pathology, 85: 15–25.
Schena Leonardo, Nigro Franco, Ippolito Antonio, Gallitelli Donato (2004): Real-time quantitative PCR: a new technology to detect and study phytopathogenic and antagonistic fungi. European Journal of Plant Pathology, 110, 893-908
SCHENA LEONARDO, HUGHES KELVIN J. D., COOKE DAVID E. L. (2006): Detection and quantification of Phytophthora ramorum, P. kernoviae, P. citricola and P. quercina in symptomatic leaves by multiplex real-time PCR. Molecular Plant Pathology, 7, 365-379
Schena L., Li Destri Nicosia M.G., Sanzani S.M., Faedda R., Ippolito A., Cacciola S.O. (2013): Development of quantitative PCR detection methods for phytopathogenic fungi and oomycetes. Journal of Plant Pathology, 95: 7–24.
Sharma Shweta, Dasgupta Indranil (2012): Development of SYBR Green I based real-time PCR assays for quantitative detection of Rice tungro bacilliform virus and Rice tungro spherical virus. Journal of Virological Methods, 181, 86-92
Su’udi Mukhamad, Kim Jinyeong, Park Jong-Mi, Bae Shin-Chul, Kim Donghern, Kim Yong-Hwan, Ahn Il-Pyung (2013): Quantification of Rice Blast Disease Progressions Through Taqman Real-Time PCR. Molecular Biotechnology, 55, 43-48
Tessitori M., Rizza S., Reina A., Catara V. (2005): Real-time RT-PCR based on Sybr-Green I for the detection of citrus exocortis and citrus cachexia disease. In: Hilf M.E., Duran-Vila N., Rocha-Peña M.A. (eds): Proceedings 16th Conference of the International Organization of Citrus Virologists, Nov 3–6, 2004. Riverside, USA: 456–459.
Tomlinson J. A., Barker I., Boonham N. (): Faster, Simpler, More-Specific Methods for Improved Molecular Detection of Phytophthora ramorum in the Field. Applied and Environmental Microbiology, 73, 4040-4047
Tsai Y.L., Olson B.H. (1991): Rapid method for direct extraction of DNA from soil and sediments. Applied and Environmental Microbiology, 57: 1070–1074.
Ward L. I., Beales P. A., Barnes A. V., Lane C. R. (2004): A Real-time PCR Assay Based Method for Routine Diagnosis of Spongospora subterranea on Potato Tubers. Journal of Phytopathology, 152, 633-638
Eden-Green Simon (2010): Phytoplasmas: Genomes, Plant Hosts and Vectors. Plant Pathology, 59, 1177-1178
Weller S.A., Beresford-Jones N.J., Hall J., Thwaites R., Parkinson N., Elphinstone J.G. (2007): Detection of Xanthomonas fragariae and presumptive detection of Xanthomonas arboricola pv. fragariae, from strawberry leaves, by real-time PCR. Journal of Microbiological Methods, 70, 379-383
Williams N., Hardy G. E. St. J., O’Brien P. A. (2009): Analysis of the distribution of Phytophthora cinnamomi in soil at a disease site in Western Australia using nested PCR. Forest Pathology, 39, 95-109
Wittwer Carl T., Herrmann Mark G., Gundry Cameron N., Elenitoba-Johnson Kojo S.J. (2001): Real-Time Multiplex PCR Assays. Methods, 25, 430-442
Wu Jinping, Diao Ying, Gu Yucheng, Hu Zhongli (2011): Molecular detection of Pectobacterium species causing soft rot of Amorphophallus konjac. World Journal of Microbiology and Biotechnology, 27, 613-618
Xu R., Tambong J. T. (2011): A TaqMan real-time PCR assay targeting the cytochrome o ubiquinol oxidase subunit II gene for detection of several pathovars of Pseudomonas syringae. Canadian Journal of Plant Pathology, 33, 318-331
Yang Jin-Guang, Wang Feng-Long, Chen De-Xin, Shen Li-Li, Qian Yu-Mei, Liang Zhi-Yong, Zhou Wen-Chang, Yan Tai-He (2012): Development of a One-Step Immunocapture Real-Time RT-PCR Assay for Detection of Tobacco Mosaic Virus in Soil. Sensors, 12, 16685-16694
Zhang Xun, Zhou Guanghe, Wang Xifeng (2010): Detection of wheat dwarf virus (WDV) in wheat and vector leafhopper (Psammotettix alienus Dahlb.) by real-time PCR. Journal of Virological Methods, 169, 416-419
Zhang Peng, Mar Thi Thi, Liu Wenwen, Li Li, Wang Xifeng (2013): Simultaneous detection and differentiation of Rice black streaked dwarf virus (RBSDV) and Southern rice black streaked dwarf virus (SRBSDV) by duplex real time RT-PCR. Virology Journal, 10, 24-
Zhao Zhe, Yu Yun, Zhang Zhixiang, Liang Pengbo, Ma Yuxin, Li Shifang, Wang Hongqing (2013): A duplex, SYBR Green I-based RT-qPCR assay for the simultaneous detection of Apple chlorotic leaf spot virus and Cherry green ring mottle virus in peach. Virology Journal, 10, 255-
Zouhar M., Mazáková J., Prokinová E., Váňová M., Ryšánek P. (2010): Quantification of Tilletia caries and Tilletia controversa mycelium in wheat apical meristem by real-time PCR. Plant Protection Science, 46: 107–115.
download PDF

© 2022 Czech Academy of Agricultural Sciences | Prohlášení o přístupnosti