Molecular Tools and Techniques for Detection and Diagnosis of Plant Pathogens

  • Pramila Pandey
  • N. S. Pandey
  • Md. Shamim
  • Deepti Srivastava
  • D. K. Dwivedi
  • L. P. Awasthi
  • K. N. Singh


Plant pathogens infect a wide range of plant species and cause great yield and quality loss of agricultural crops. Detection and accurate identification of harmful plant pathogens is very essential to improve the strategies for controlling plant diseases. The early detection and identification of plant pathogens provides the basis for understanding their biology and appropriate strategies to control that particular pathogen. For the identification of plant pathogen, traditional procedures, i.e., isolation, in vitro culturing and microscopy of the extracellular pathogens, are in common routine. However, traditional methods may take days or weeks for particular pathogens to produce diagnostic spores. Indexing for many intracellular pathogens is also very complex because they are obligate biotrophs in nature. The development in the recent tools in molecular biology has enhanced and accelerated the detection and diagnostics through the automatic purification of nucleic acids and specific proteins from pathogens.


Polymerase Chain Reaction Plant Pathogen Cucumber Mosaic Virus Multiplex Polymerase Chain Reaction Nest Polymerase Chain Reaction 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. Adaskaveg JE, Hartin RJ (1997) Characterization of Colletotrichum acutatum isolates causing anthracnose of almond and peach in California. Phytopathology 87:979–987CrossRefPubMedGoogle Scholar
  2. Agrios GN (2001) Fitopatologia, 2nd edn. Uteha Noriega Editores, MexicoGoogle Scholar
  3. Allen WR, Matteoni JA (1991) Petunia as an indicator plant for use by growers to monitor for thrips carrying the tomato spotted wilt virus in greenhouses. Plant Dis 75:78–82CrossRefGoogle Scholar
  4. Alvarez AM (2001) Differentiation of bacterial populations in seed extracts by flow cytometry. In: de Boer SH (ed) Plant pathogenic bacteria. Kluwer, Dordrecht, pp 393–396CrossRefGoogle Scholar
  5. Bariana HS, Shannon AL, Chu PWG, Waterhouse PM (1994) Detection of five seedborne legume viruses in one sensitive multiplex polymerase chain reaction test. Phytopathology 84:1201–1205CrossRefGoogle Scholar
  6. Beck JJ, Ligon JM, Etienne L, Binder A (1996) Detection of crop fungal pathogens by polymerase chain reaction technology. In: Marshall G (ed) Diagnostics in crop production, BCPC symposium proceedings No 65. British Crop Protection Council, Farnham, pp 111–118Google Scholar
  7. Becker H, Manz A (1999) Microsystem technology in chemistry and life science. Springer Desktop Editions in Chemistry, vol 194. Springer, Berlin/Heidelberg/New YorkGoogle Scholar
  8. Bertaccini A (1990) Detection of chrysanthemum yellows mycoplasmalike organism by dot hybridization and Southern blot analysis. Plant Dis 74:40–43CrossRefGoogle Scholar
  9. Bertolini E, Olmos A, Martınez MC, Gorris MT, Cambra M (2001) Single-step multiplex RT-PCR for simultaneous and colourimetric detection of six RNA viruses in olive trees. J Virol Methods 96:33–41CrossRefPubMedGoogle Scholar
  10. Bertolini E, Olmos A, Lopez MM, Cambra M (2003) Multiplex nested reverse-transcription polymerase chain reaction in a single tube for sensitive and simultaneous detection of four RNA viruses and Pseudomonas savastanoi pv. savastanoi in olive trees. Phytopathology 93:286–292CrossRefPubMedGoogle Scholar
  11. Biosca EG, Marco-Noales E, Ordax M, Lopez MM (2006) Long-term starvation-survival of Erwinia amylovora in sterile irrigation water. Acta Horticult 704:107–112CrossRefGoogle Scholar
  12. Bird RE, Hardman KD, Jacobson JW, Johnson S, Kaufman BM, Lee S, Lee T, Pope SH, Riordan GS, Whitlow M (1988) Single-chain antigen-binding proteins. Science 242:423–426CrossRefPubMedGoogle Scholar
  13. Bock KR (1982) The identification and partial characterization of plant-viruses in the tropics. Trop Pest Manag 28:399–411CrossRefGoogle Scholar
  14. Bonde MR, Micales JA, Peterson GL (1993) The use of isozyme analysis for identification of plant-pathogenic fungi. Plant Dis 77:961–968CrossRefGoogle Scholar
  15. Cahill DM, Hardham AR (1994) Exploitation of zoospore taxis in the development of a novel dipstick immunoassay for the specific detection of Phytophthora cinnamomi. Phytopathology 84:193–200CrossRefGoogle Scholar
  16. Cambra M, Capote N, Myrta A, Llacer G (2006) Plum pox virus and the estimated costs associated with sharka disease. Bull OEPP/EPPO 36:202–204CrossRefGoogle Scholar
  17. Caruso P, Bertolini E, Cambra M, Lopez MM (2003) A new and co-operational polymerase chain reaction (Co-PCR) for rapid detection of Ralstonia solanacearum in water. J Microbiol Methods 55(1):257–272Google Scholar
  18. Chandelier A, Dubois N, Baelen F, De Leener F, Warnon S, Remacle J, Lepoivre P (2001) RT-PCR, ELISA tests on pooled sample units for the detection of virus Y in potato tubers. J Virol Methods 91:99–108Google Scholar
  19. Chittaranjan S, De Boer SH (1997) Detection of Xanthomonas campestris pv. pelargonii in geranium and greenhouse nutrient solution by serological and PCR techniques. Eur J Plant Pathol 103:555–563CrossRefGoogle Scholar
  20. Cottyn B (1996) Bacterial diseases of rice. characterization of pathogenic bacteria associated with sheath rot complex and grain discoloration of rice in the Philippines. Plant Dis 80:438–445CrossRefGoogle Scholar
  21. Damsteegt VD (1997) Prunus tomentosa as a diagnostic host for detection of plum pox virus and other Prunus viruses. Plant Dis 81:329–332CrossRefGoogle Scholar
  22. Davey HM, Kell DB (1996) Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single cell analyses. Microbiol Rev 60:641–696PubMedCentralPubMedGoogle Scholar
  23. De Laat PCA, Verhoeven JTW, Janse JD (1994) Bacterial leaf rot of Aloe vera L, caused by Erwinia chrysanthemi biovar-3. Eur J Plant Pathol 100:81–84CrossRefGoogle Scholar
  24. Duffy BK, Weller DM (1994) A semiselective and diagnostic medium for Gaeumannomyces graminis var tritici. Phytopathology 84:1407–1415CrossRefGoogle Scholar
  25. Fatmi M, Schaad NW (1988) Semiselective agar medium for isolation of Clavibacter michiganense subsp. michiganensis from tomato seed. Phytopathology 78:121–126Google Scholar
  26. Fatmi M, Schaad NW, Bolkan HA (1991) Seed treatments for eradicating Clavibacter michiganensis subsp. michiganensis from naturally infected tomato seeds. Plant Dis 75:383–385CrossRefGoogle Scholar
  27. Fillhart RC, Bachand GD, Castello JD (1998) Detection of infectious tobamoviruses in forest soils. Appl Environ Microbiol 64:1430–1435PubMedCentralPubMedGoogle Scholar
  28. Fraaije BA, Lovell DJ, Rohel EA, Hollomon DW (1999) Rapid detection and diagnosis of Septoria tritici epidemics in wheat using a polymerase chain reaction PicoGreen assay. J Appl Microbiol 86:701–708CrossRefGoogle Scholar
  29. Fraaije BA, Lovell D, Coelho JM, Baldwin S, Hollomon DW (2001) PCR-based assays to assess wheat varietal resistance to blotch (Septoria tritici and Stagonospora nodorum) and rust (Puccinia striiformis and Puccinia recondita) diseases. Eur J Plant Pathol 107:905–917CrossRefGoogle Scholar
  30. Fraaije BA, Butters JA, Coelho JM, Johes DR, Hollomon DW (2002) Following the dynamics of strobilurin resistance in Blumeria graminis f. sp. tritici using quantitative allele-specific realtime PCR measurements with the fluorescent dye SYBR green I. Plant Pathol 51:45–54CrossRefGoogle Scholar
  31. Gilliland G, Perrin S, Blanchard K, Bunn HF (1990) Analysis of cytokine mRNA and DNA: detection and quantization by competitive polymerase chain reaction. Proc Natl Acad Sci U S A 87:2725–2729PubMedCentralCrossRefPubMedGoogle Scholar
  32. Gitaitis R et al (1997) Bacterial streak and bulb rot of onion.1. A diagnostic medium for the semiselective isolation and enumeration of Pseudomonas viridiflava. Plant Dis 81:897–900CrossRefGoogle Scholar
  33. Goor MC et al (1984) The use of API systems in the identification of phytopathogenic bacteria. Mededelingen Van De Faculteit Landbouwtenschappen Rijksuniversiteit. Genetics 49:499–507Google Scholar
  34. Grey BE, Steck TR (2001) The viable but non culturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection. Appl Environ Microbiol 67:3866–3872PubMedCentralCrossRefPubMedGoogle Scholar
  35. Griep RA et al (1998) Development of specific recombinant monoclonal antibodies against the lipopolysaccharide of Ralstonia solanacearum race 3. Phytopathology 88:795–803CrossRefPubMedGoogle Scholar
  36. Gurtler V, Stanisich VA (1996) New approaches to typing and identification of bacteria using the 16S–23S rDNA spacer region. Microbiology 142:3–16CrossRefPubMedGoogle Scholar
  37. Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH (1997) Multiplex PCR: critical parameters and step-by step protocol. Biotechniques 23:504–511PubMedGoogle Scholar
  38. Hodgson RAJ, Wall GC, Randles JW (1998) Specific identification of coconut tinangaja viroid for differential field diagnosis of viroids in coconut palm. Phytopathology 88:774–781CrossRefPubMedGoogle Scholar
  39. James D (1999) A simple and reliable protocol for the detection of apple stem grooving virus by RT-PCR and in a multiplex PCR assay. J Virol Methods 83:1–9CrossRefPubMedGoogle Scholar
  40. Janse JD (2007) Phytobacteriology: principles and practice. CABI Publishing, WallingfordGoogle Scholar
  41. Janse JD, Wenneker M (2002) Possibilities of avoidance and control of bacterial plant diseases when using pathogen-tested (certified) or -treated planting material. Plant Pathol 51:523–536CrossRefGoogle Scholar
  42. Janse J, Van den Beld HE, Elphinstone J, Simpkins S, Tjou-Tam-Sin NNA, Van Vaerenbergh J (2004) Introduction to Europe of Ralstonia solanacearum biovar 2, race 3 in Pelargonium zonale cuttings. J Plant Pathol 86:147–155Google Scholar
  43. Jeffers SN, Aldwinckle HS (1987) Enhancing detection of Phytophthora cactorum in naturally infested soil. Phytopathology 77:1475–1482CrossRefGoogle Scholar
  44. Jones DAC (1994) Blackleg potential of potato seed – determination of tuber contamination by Erwinia carotovora subsp atroseptica by immunofluorescence colony staining and stock and tuber sampling. Ann Appl Biol 124:557–568CrossRefGoogle Scholar
  45. Kittipakorn K et al (1993) Strains of peanut stripe potyvirus rapidly identified by profiling peptides of the virion proteins. J Phytopathol Phytopathologische Z 137:257–263CrossRefGoogle Scholar
  46. Knoll S, Vogel RF, Niessen L (2002) Identification of Fusarium graminearum in cereal samples by DNA Detection Test Strips. Lett Appl Microbiol 34:144–148CrossRefPubMedGoogle Scholar
  47. Landgraf A, Reckmann B, Pingoud A (1991) Direct analysis of polymerase chain reaction products using enzyme-linked immunosorbent assay techniques. Ann Biochem 198:86–91CrossRefGoogle Scholar
  48. Lopez MM, Bertolini E, Olmos A, Caruso P, Gorris MT, Llop P, Penyalver R, Cambra M (2003) Innovative tools for detection of plant pathogenic viruses and bacteria. Int Microbiol 6:233–243CrossRefPubMedGoogle Scholar
  49. Lopez MM, Bertolini E, Marco-Noales E, Llop P, Cambra M (2006) Update on molecular tools for detection of plant pathogenic bacteria and viruses. In: Rao JR, Fleming CC, Moore JE (eds) Molecular diagnostics: current technology and applications. Horizon Bioscience, Wymondham, pp 1–46Google Scholar
  50. Louws FJ, Rademaker JLW, de Bruijn FJ (1999) The three Ds of PCR-based genomic analysis of phytobacteria: diversity, detection, and disease diagnosis. Annu Rev Phytopathol 37:81–125CrossRefPubMedGoogle Scholar
  51. Manandhar JB, Hartman GL, Wang TC (1995) Semiselective medium for Colletotrichum gloeosporioides and occurrence of 3 Colletotrichum spp on pepper plants. Plant Dis 79:376–379CrossRefGoogle Scholar
  52. Martin RR, James D, Levesque CA (2000) Impacts of molecular diagnostic technologies on plant disease management. Annu Rev Phytopathol 38:207–239CrossRefPubMedGoogle Scholar
  53. Miller SA, Martin RR (1988) Molecular diagnosis of plant disease. Annu Rev Phytopathol 26:409–432CrossRefGoogle Scholar
  54. Milne RG, Ramasso GE, Lenzi R, Masenga V, Sarindu S, Clark MF (1995) Pre- and post-embedding immunogold labeling and electron microscopy in plant host tissues of three antigenically unrelated MLOs: primula yellows, tomato big bud and bermudagrass whiteleaf. Eur J Plant Pathol 10:57–67CrossRefGoogle Scholar
  55. Mullis KB, Faloona FA (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155:335–350CrossRefPubMedGoogle Scholar
  56. Mutasa ES, Ward E, Adams MJ, Collier CR, Chwarszczynska D, Asher MJC (1993) A sensitive DNA probe for the detection of Polymyxa betae in sugar beet roots. Physiol Mol Plant Pathol 43:379–390CrossRefGoogle Scholar
  57. Mutasa ES, Chwarszczynska D, Adams MJ, Ward E, Asher MJC (1995) Development of PCR for the detection of Polymyxa betae in sugar beet roots and its application in field studies. Physiol Mol Plant Pathol 47:303–313CrossRefGoogle Scholar
  58. Mutasa ES, Chwarszczynska DM, Asher MJC (1996) Single-tube, nested PCR for the diagnosis of Polymyxa betae infection in sugar beet roots and colorimetric analysis of amplified products. Phytopathology 86:493–497CrossRefGoogle Scholar
  59. Nassuth A, Pollari E, Helmeczy K, Stewart S, Kofalvi SA (2000) Improved RNA extraction and one-tube RT-PCR assay for simultaneous detection of control plant RNA plus several viruses in plant extracts. J Virol Methods 90:37–49CrossRefPubMedGoogle Scholar
  60. Nicholson P, Lees A, Maurin N, Parry D, Rezanoor HN (1996) Development of a PCR assay to identify and quantify Microdochium nivale var. nivale and Microdochium nivale var. majus in wheat. Physiol Mol Plant Pathol 48:257–271Google Scholar
  61. Nicholson P, Simpson DR, Weston G, Rezanoor HN, Lees AK, Parry DW, Joyce D (1998) Detection and quantification of Fusarium culmorum and Fusarium graminearum in cereals using PCR assays. Physiol Mol Plant Pathol 53:17–37CrossRefGoogle Scholar
  62. Okuda M, Hanada K (2001) RT-PCR for detecting five distinct Tospovirus species using degenerate primers and dsRNA template. J Virol Methods 96:149–156CrossRefPubMedGoogle Scholar
  63. Olmos A, Cambra M, Esteban O, Gorris MT, Terrada E (1999) New device and method for capture, reverse transcription and nested PCR in a single closed-tube. Nucleic Acids Res 27:1564–1565PubMedCentralCrossRefPubMedGoogle Scholar
  64. Olmos A, Bertolini E, Cambra M (2002) Simultaneous and cooperational amplification (Co-PCR): a new concept for detection of plant viruses. J Virol Methods 106:51–59CrossRefPubMedGoogle Scholar
  65. Olmos A, Esteban O, Bertolini E, Cambra M (2003) Nested RT-PCR in a single closed tube. In: Bartlett JMS, Stirling D (eds) Methods in molecular biology, vol 226, 2nd edn, PCR Protocols. Humana Press, Ottowa, pp 156–161Google Scholar
  66. Pernezny K (1995) An outbreak of bacterial spot of lettuce in Florida caused by Xanthomonas campestris pv. vitians. Plant Dis 79:359–360CrossRefGoogle Scholar
  67. Piotte C (1994) Cloning and characterization of 2 satellite DNAs in the low-C-value genome of the nematode Meloidogyne spp. Gene 138:175–180Google Scholar
  68. Piotte C (1995) Analysis of a satellite DNA from Meloidogyne hapla and its use as a diagnostic probe. Phytopathology 85:458–462Google Scholar
  69. Rasmussen OF, Reeves JC (1992) DNA probes for the detection of plant pathogenic bacteria. J Biotechnol 25:203–220CrossRefGoogle Scholar
  70. Rasmussen JB, Scheffer RP (1988a) Effects of selective toxin from Helminthosporium carbonum on chlorophyll synthesis in maize. Physiol Mol Plant Pathol 32:283–291CrossRefGoogle Scholar
  71. Rasmussen JB, Scheffer RP (1988b) Isolation and biological activities of four selective toxins from Helminthosporium carbonum. Plant Physiol 86:187–191PubMedCentralCrossRefPubMedGoogle Scholar
  72. Roberts PD (1996) Survival of Xanthomonas fragariae on strawberry in summer nurseries in Florida detected by specific primers and nested polymerase chain reaction. Plant Dis 80:1283–1288CrossRefGoogle Scholar
  73. Roy MA (1988) Use of fatty-acids for the identification of phytopathogenic bacteria. Plant Dis 72:460Google Scholar
  74. Russo P, Miller L, Singh RP, Slack SA (1999) Comparison of PLRV and PVY detection in potato seed samples tested by Florida winter field inspection and RT-PCR. Am J Potato Res 76:313–316CrossRefGoogle Scholar
  75. Saade M, Aparicio F, Sanchez-Navarro JA, Herranz MC, Di-Terlizzi AMB, Pallas V (2000) Simultaneous detection of the three ilarviruses affecting stone fruit trees by nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction. Phytopathology 90:1330–1336CrossRefPubMedGoogle Scholar
  76. Schaad NW, Frederick RD (2002) Real-time PCR and its application for rapid plant disease diagnostics. Can J Plant Pathol 24:250–258CrossRefGoogle Scholar
  77. Schots A, Dewey FM, Oliver R (eds) (1994) Modern assays for plant pathogenic fungi: identification, detection and quantification. CAB International, WallingfordGoogle Scholar
  78. Scortichini M (1995) Le malattie batteriche delle colture agrarie e delle specie forestali. Edagricole, Edizione Agricole, BolognaGoogle Scholar
  79. Shurtleff MC, Averre CW III (1997) Glossary of plant-pathological terms. American Phytopathology Association, St. PaulGoogle Scholar
  80. Singh RP, Kurz J, Boiteau G (1996) Detection of stylet-borne and circulative potato viruses in aphids by duplex reverse transcription polymerase chain reaction. J Virol Methods 59:189–196CrossRefPubMedGoogle Scholar
  81. Strange RN (1993) Plant disease control: towards environmentally acceptable methods, using PCR assays. Physiol Mol Plant Pathol 53:17–37Google Scholar
  82. Van der Wolf JM, Van Beckhoven JRCM, Bonanats PJM, Schoen CD (2001) New technologies for sensitive and specific routine detection of plant pathogenic bacteria. In: de Boer SH (ed) Plant pathogenic bacteria. Kluwer, Dordrecht, pp 75–77CrossRefGoogle Scholar
  83. Van Dijk P, Vandermeer FA, Piron PGM (1987) Accessions of Australian Nicotiana species suitable as indicator hosts in the diagnosis of plant virus diseases. Neth J Plant Pathol 93:73–85CrossRefGoogle Scholar
  84. Van Sluys MA, Monteiro-Vitorello CB, Camargo LEA, Menck CFM, da Silva ACR, Ferro JA, Oliveira MC, Setubal JC, Kitajima JP, Sympson AJ (2002) Comparative genomic analysis of plant-associated bacteria. Ann Rev Phytopathol 40:169–190CrossRefGoogle Scholar
  85. Volkhard A, Kempf J, Trebesius K, Autenrieth IB (2000) Fluorescent in situ hybridization allows rapid identification of microorganisms in blood cultures. J Clin Microbiol 38:830–838Google Scholar
  86. Wang AM, Doyle MV, Mark DF (1989) Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci U S A 24:9717–9721CrossRefGoogle Scholar
  87. Ward E, Gray R (1992) Generation of a ribosomal DNA probe by PCR and its use in identification of fungi within the Gaeumannomyces-Phialophora complex. Plant Pathol 41:730–736CrossRefGoogle Scholar
  88. Wells JM, Vanderzwet T, Butterfield JE (1994) Differentiation of Erwinia species in the herbicola group by class analysis of cellular fatty-acids. J Phytopathol Phytopathol Z 140:39–48CrossRefGoogle Scholar
  89. Williams K, Blake S, Sweeney A, Singer JT, Nicholson BL (1999) Multiplex reverse transcriptase PCR assay for simultaneous detection of three fish viruses. J Clin Microbiol 37:4139–4141PubMedCentralPubMedGoogle Scholar
  90. Williams R, Ward E, McCartney HA (2001) Methods for integrated air sampling and DNA analysis for detection of airborne fungal spores. Appl Environ Microbiol 67:2453–2459PubMedCentralCrossRefPubMedGoogle Scholar
  91. Winter G, Griffiths AD, Hawkins RE, Hoogenboom HR (1994) Making antibodies by phage display technology. Ann Rev Immunol 12:433–455CrossRefGoogle Scholar
  92. Wullings BA, Beuningen AR, Van Janse JD, Akkermans ADL, Van Beuningen AR (1998) Detection of Ralstonia solanacearum, which causes brown rot of potato, by fluorescent in situ hybridization with 23S rRNA-targeted probes. Appl Environ Microbiol 64:4546–4554PubMedCentralPubMedGoogle Scholar
  93. Zeringue HJ, Bhatnagar D, Cleveland TE (1993) C15H24 volatile compounds unique to aflatoxigenic strains of Aspergillus flavus. Appl Environ Microbiol 59:2264–2270PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer India 2015

Authors and Affiliations

  • Pramila Pandey
    • 1
  • N. S. Pandey
    • 2
  • Md. Shamim
    • 3
    • 4
  • Deepti Srivastava
    • 1
  • D. K. Dwivedi
    • 1
  • L. P. Awasthi
    • 4
  • K. N. Singh
    • 1
  1. 1.Department of Plant Molecular Biology and Genetic EngineeringN.D. University of Agriculture and TechnologyFaizabadIndia
  2. 2.Department of BotanyNational P.G. CollegeLucknowIndia
  3. 3.Department of Molecular Biology and Genetic EngineeringBihar Agricultural UniversitySabour BhagalpurIndia
  4. 4.Department of Plant PathologyN.D. University of Agriculture and TechnologyFaizabadIndia

Personalised recommendations