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Current methods for plant disease diagnosis
- 2. Current and Prospective Methodsfor Plant Disease Detection Speaker : Shivani PhD 2nd Year 48020 Doctoral Seminar-I on
- 3. OUTLINE Introduction Serological based detectionmethods Nucleic acid based detection methods Optical sensors based detection methods Digital imaging based detection methods Biosensors based detection methods 3 Case studies Conclusion Future Prospects
- 4. Importance Effective cropmanagement and regulatory programmes Determination of cause, epidemiology and distribution of diseases Providing suitable plant protection measures Resolving the components of complex diseases Seed certification and quarantine Studying taxonomic and evolutionary relationship of plant pathogens Detection and identification of new pathogens Assessment of disease incidence and yield loss in field 4
- 5. Implications Risk ofmovement of pathogens and their vectors from one country to another Preventing outbreaks and potentially devastating crop diseases Identifying variability of the organism To detect pathogens before symptom development To screen large number of samples accurately, reliably, quickly with greater sensitivity To find the ways that provide additional information Improvements 5
- 6. Current diagnostic methodsfor plant disease detection Use of recombinant antibodies Serological tests • R-ELISA • Lateral flow devices Micro-arrays (Bio-chips) Nucleic acid based tests • Real-time PCR • Nested and Multiplex PCR Digital Consulting System or imaging FISH Flow Cytometry Thermography Hyperspectral techniques Gas Chromatography 6
- 7. 7 Serological Based detectionTechniques
- 8. Polyclonal antibodies(Pabs) Demerits: Cross-reactions to contaminants Lack of sustainable source of Abs Lack of specificity Monoclonal antibodies (Mabs) Superior to Pabs as can provide constant supply of specific antibodies Expensive to produce and maintain Recombinant antibodies Phage expressing complete Ab variable domain Commercial available libraries can be screened with plant pathogen to identify clones Clones formed are used as a source of Abs for a diagnostic system e.g. Potato leaf roll virus, Tomato spotted wilt virus, Alternaria alternata, Ralstonia solanacearum, Phytophthora infestans, Cucumber mosaic virus (Zienglar and Torrance, 2002) Advantages No need to immunize any animal Monovalency Rapid, sensitive and low cost Potential to be used for a wide range of plant pathogens 8
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- 10. ELISA as diagnostictools Viruses Tomato spotted wilt virus , Citrus tristeza virus, Groundnut viruses, Rice tungro virus, Rice grassy stunt virus, Maize steak virus, Banana bunchy top , Papaya ring spot , Sugarcane mosaic , Maize dwarf, Poty viruses, Tospo viruses, All potato viruses, Tobra virus, Tobacco ring spot virus, Alfa-alfa mosaic virus Fungi Phytophthora infestans, Pythium spp., Rhizoctonia spp, Septoria tritici, Septoria nodorum, Colletotrichum spp., Monilinia fructicola, Botrytis cinerea, Sclerotinia spp., P. graminis, Fusarium spp. Bacteria Ralstonia solanacearum, Clavibacter michiganensis sub sp. sepidonicus and michiganensis, Erwinia carotovora, Erwinia chrysanthemi, Xanthomonas campestris pv. oryzae Nematodes Globodera pallida, G.rostochiensis, Meloidogyne spp. Others Spiroplasma citri 10
- 11. Pros and consof using ELISA Advantages Simple, Specific, accurate and quick Little expertise required Detection of pathogens with variable or latent symptoms Disadvantages Not effective for viroids High initial cost 11
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- 13. Limitations Ideal forviruses and bacteria (symptomatic material) Limited use for fungi (difficulty to achieve species specificity) Life stage specific 13
- 14. Nucleic acid Baseddetection Techniques
- 15. Polymerase Chain Reaction(PCR) Most important technique in molecular biology Amplifies specific DNA sequence exponentially, by chain reaction 15
- 16. Classical PCRhas not been adopted by most plant disease regulatory and diagnostic labs Why ? Give results of PCR test in real time As a diagnostic technology, it is remarkably: • Rapid • Sensitive • Specific Real time or quantitative PCR 16
- 17. Detection andquantification of a fluorescent transmitter during the process of amplification Increase in the fluorescent signal is directly proportional to the quantity of amplicon produced during the reaction Hybridization of fluorescently labeled oligonucletide probe sequences Specific region within the target amplicon Amplification using traditional forward and reverse PCR primers • Probe hydrolysis (Taqman probe) 17
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- 19. Advantages of Real TimePCR Time saving (no gel- running) Closed-tube system (reduced contamination risk) Easy multiplexing (internal controls) Generic assays Increased sensitivity Quantitation 19
- 20. Real time PCRassays in diagnostics R. solanacearum, Agrobacterium, Xylella fastidiosa, C. sepedonicus, Erwinia, Acidovorax avenae subsp. citrulli, Pierce’s disease Tilletia indica, Phakospora pachyrhizi, Phytophthora infestans, P. citricola, Diaporthe phaseolorum,Phomosis longicola, Helmintosporium solani, Pyrenophora graminea Tomato spotted wilt virus, Sugarcane yellow leaf virus, Potato mop-top virus,Tobacco rattle virus, Cymbidium mosaic potyvirus, odontoglossum ringspot tobamo virus Potato cyst nematode (Schaad & Frederick, 2002, Schaad et al., 2003 20
- 21. PCR canbe used in combination with pathogen isolation on agar media Using a combine viable enrichment (growth media) with an enzymatic amplification (PCR) Target bacterium can be enriched in liquid or solid media and detected extremely low levels in seeds and other propagative materials • Increased sensitivity • Elimination of PCR inhibition • Detection of viable cell only BIO-PCR 21 Extracting a sample Plating a sample onto agar media Incubating for 15-72 h Washing plates and centrifugation 1 or 10 µL for direct PCR
- 22. Nested PCR A secondround of PCR is performed with amplified DNA as template and primers internal to first round primers Multiplex PCR Allows the simultaneous and senstivity detection of different DNA or RNA targets in a single reaction Multiplex nested PCR combines both multiplex and nested PCR 22
- 23. 23 Single site templatePCR Multiple site template PCR
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- 25. Nested PCR inplant pathology Pythium ultimum, Plasmodiophora brassicae, Verticillium dahliae Clavibacter michiganensis subsp. sepedonicus, Xanthomonas axonopodis pv. manihotis ,Erwinia amylovora, Pseudomonas savastanoi pv. Savastanoi, X. fastidiosa Cucumber mosaic virus, Cherry leaf roll virus, Strawberry latent ring spot virus, Arabis mosaic virus (Pabla et al., 2000; Edson et al., 2002 ;Ojeda & Verdier, 2005) Limitations False positive reaction due to contamination Cost of test is high Additional cost of reverse transcriptase for cDNA in case of plant viruses 25
- 26. Micro-arrays (Bio-chip) Array:orderlyarrangement of samples Medium for matching known and unknown NA samples Generated by depositing a few nanolitres of DNA on a solid support Developed for high throughout measurement expression patterns of thousands of genes Principle: Base pairing of complementary sequences by hybridization 26
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- 28. Loop mediated isothermalamplification Loop-mediated Refers to the loop structures formed when the LAMP primers amplify their target DNA sequences Isothermal The reaction takes place at single temperature (63- 67oC Amplification The highest efficient polymerase enzyme used amplifies the very small amount of target in the sample, generating millions of the copies of the sequence LAMP 28 LAMP is used in rapid diagnosis of viral and bacterial plant pathogens
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- 31. PCR based techniques Genomicassay Targeted taxa References PCR Agrobacterium tumefaciens Candidatus Liberobacter, E. carotovora Tospo &Citrus Tristeza virus Tilletia indica Cubero et al 2002, Ahlawat et al., 2004, Dorasse et al ., 2003 Okuda et al., 2000, Ramachandran et al., 2002 Frederick et al., 2000 Co-PCR R. solanacearum Caruso et al., 2003 Multiplex nested PCR Pseudomonas savastanoi pv. savastanoi Begomo virus Bertolini et al., 2003 Potter et al, 2003 Multiplex PCR Xiphinema index Globodera rostochiensis, G. pallida Cucumovirus, Nepovirus Necrovirus, Olea virus Wang et al., 2002 Mulholand et al., 2001 Bertolini et al., 2001 Nested PCR X. axonopodis pv. citri, P. savastanoi pv. savastanoi Hartung et al. (1996) (Bertolini et al., 2003) 31
- 32. 32 FISH (Fluorescence InSitu Hybridization)
- 33. Applied for bacterialdetection in combination with microscopy and hybridization of DNA probes and target gene from plant samples Due to the presence of pathogen specific rRNA sequences in plants Advantage of high sensitivity (103 CFU/mL) Could also be used to detect fungi and viruses and other endosymbiotic bacteria that infect the plant 33 This technique could also be used to detect unculturable or yet-to-be cultured organisms to investigate complex microbiome
- 34. Indian Scenario DAS- ELISA DAC-ELISA DIBA PNC-ELISA R-PAGE Lily carla virus, Carnation mottle virus, Bean yellow mosaic, Groundnut bud necrosis virus, CMV, Banana streak virus, Water melon mosaic virus, Stripe teniuvirus in Fodder, Viroids, TSV Bhaik et al 2004, Singh et al 2004, Roy et al 2005, Raja & Jain, 2006,Bhat et al 2004, Bhadramurthy et al 2005, Biswas &Verma 2005, Narayana 2004, Suryanarayana et al 2004 TEM (leaf dip serology) ISEM Watermelon mosaic virus, Indian citrus ringspot virus Biswas &Verma 2005 Hoa & Ahlawat 2004 RT-PCR Groundnut bud necrosis virus, Indian citrus ringspot virus, Banana streak virus, Citrus yellow mosaic virus in P. citri, Raja & Jain 2006, Hoa & Ahlawat 2004, Raman et al 2004, Saxena et al 2005 Singh et al 2004 PCR (ITS) R. solanacearum , C.gloesporioides & falcatum U.scitaminea Kumar & Anandaraj 2006, Pandey & Pandey 2006, Naik & Gaikwad 2005 PCR (16s rRNA) Grassy shoot of sugarcane Srivastava et al 2004 Nested PCR GSD and yellow leaf syndrome of sugarcane Rao et al 2004 PCR-RFLP RTV,X. axonopodis pv. malvacearum, CLCuV Niazi et al 2005, Chakraborthy et al 2004, Microsatelite markers Fusarium spp. Prasad et al 2004 34
- 35. Optical sensors baseddetection Techniques
- 36. FCM (Flow cytometry) Flowcytometry (FCM) is a laser- based optical technique widely used for cell counting and sorting, biomarker detection and protein engineering The technique uses an incident laser beam and measures the scattering and fluorescence of the laser beam reflected from the sample FCM is used for rapid identification of cells while cells pass through an electronic detection apparatus in a liquid stream (Chitarra et al., 2003) 36
- 37. Thermography Allows imaging thedifferences in surface temperature of plant leaves and canopies Emitted infrared radiation can be captured by thermographic cameras and color difference can be analyzed Also a promising tool to monitor the heterogenity in the infection of soilborne pathogens The practical applicability of thermography for disease monitoring is limited due to its high sensitivity to the change of environmental conditions during measurement (Chaerle, et al., 2007) 37
- 38. Fluorescence imaging The chlorophyllfluorescence is measured on the leaves as a function of the incident light and the change in fluorescence parameters can be used to analyze pathogen infections, based on changes in the photosynthetic apparatus and photosynthetic electron transport reactions Temporal and spatial variations of chlorophyll fluorescence were analyzed for precise detection of leaf rust and powdery mildew infections in wheat leaves at 470 nm (Kuchenberg, et al., 2009) 38 The practical application of this technique in a field setting is limited
- 39. Hyperspectral techniques Used toobtain useful information about the plant health over a wide range of spectrum between 350 and 2500 nm Highly robust and it provides a rapid analysis of the imaging data Magnaporthe grisea infection of rice, Phytophthora infestans infection of tomato and Venturia inaequalis infection of apple trees have been identified and reported using hyperspectral imaging techniques (Kobayashi, et al., 2007) 39
- 40. 40 How does hyperspectralimaging work • Hyperspectral imaging deals with the imaging of narrow spectral bands over a continuous spectral range, and produces the spectra of all pixels in the scene • Hyperspectral sensors collect information as a set of “images” • These images are then combined and formed into three-dimensional hyperspectral data cube for processing and analysis
- 41. Gas chromatography A completelydifferent non- optical indirect method for plant disease detection involves the profiling of the volatile chemical signature of the infected plants An infection by Phytophthora cactorum, the fungus that causes crown rot diseases in strawberries, results in the release of p- ethylguaiacol and p-ethylphenol as characteristic VOCs from the infected portion of the strawberry plant/fruit (Fang et al., 2014) 41
- 42. The volatile signatureof plants could be analyzed using gas- chromatography (GC) technique to analyze the presence of the specific VOC that is indicative of a particular disease It also allows the detection of diseases at different stages based on the quantitative information collected from the VOC sample 42
- 43. VOCs emitted fromwhole, intact tomato plants or detached leaves, and biotic stress causing agents responsible for increase in VOC emissions 43
- 44. 44 Digital imaging baseddtection techniques
- 45. Digital imaging system Distant analysis can be used in first line diagnostic Can save a lot of time Better and faster communication between inspectors and specialists Sharing of knowledge Developing central database Always final identification by diagnostic specialist 45
- 46. Imaging techniques Use Magneticresonance microscopy (MRM) Pine wilt disease (Utsuzawa et al., 2005 ) Atomic force microscopy (AFM) Tobacco mosaic viruses X-Ray Spectroscopy Mass spectrometry based proteomics Rhizoctonia solani, Fusarium graminearum, and Ustilago maydis, Uromyces appendiculatus (Padliya and Copper, 2006) 46
- 47. Ideal diagnostic strategy High detectability Specificity Sensitivity Accuracy supported with simplicity Amenable to automation Low cost Issues related to diagnostics Cost Specificity Sensitivity 47
- 48. 48 Biosensors based detectionTechniques
- 49. 49 How does biosensorswork? Analyte of interest Biorecepter Transducer Signal Detection of plant diseases using portable sensors Antibody Enzymes Phages Oligonucleotides Optical Electrochemi cal Mechanical Cantilever Luminescence Fluorescence UV-VS Magnetic Resonance
- 50. Biosensor platforms basedon nanomaterials Affinity biosensors Antibody-based biosensors DNA/RNA-based affinity biosensor Enzymatic electrochemical biosensors Bacteriophage-based biosensors 50 Types of biosensors (Singh et al., 2010)
- 51. Comparison of affinitybiosensors, enzymatic biosensors, nanomaterials modified sensors and ELISA 51
- 52. List of commerciallyavailable sensors for plant fungal detection Manufacturer Product Principle Pathogens BIOREBA Agristrip Lateral flow immunochromatography Spongospora subterranea Pocket Diagnostic Pocket Diagnostic Kits Lateral flow assay Phytophthora Neogen Corporation Alert Test Kits Lateral flow assay Pythium, Phytophthora, Rhizoctonia Agdia Immunostrip Lateral flow immunochromatography Pythium, Phytophthora La Chandra Bioscience Pocket Diagnostic test kits Lateral flow technology Pythium, Phytophthora, Rhizoctonia solani, Botrytis Cepheid Smart Cycler Real Time PCR Aspergillus, Phalospora sp. Fera Portable PCR RT PCR Phytophthora ramorum Ahram Biosystems Palm PCR RT PCR Aspergillus Electronic Sensors Technology zNose 4200, 4300,7100 Electronic nose (E-nose) Oidium neolycopersici Sensigent Cyranose 320 E-nose Botrytis cineria, Colletotrichum gleosporioides, Alternaria sp. Sensitive Technologies Limited Bloodbound ST214 E-nose Oidium neolycopersici 52
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- 56. Multispectral imaging system(MSIS) developed for sunlight-induced fluorescence imaging of outdoor plants 56 *EMCCD: Electron multiplying charge coupled device Non-destructive method for detection and classification of CMD infection based on far:far-red chlorophyll fluorescence image ratio Study was carried out in 14 varieties of potted cassava plant with a MSIS consisting of an EMCCD camera Sunlight induced chl fluorescence (SICF) images of plants were recorded using the MSIS at different wavelength The red: far red ratio was computed and correlated with the laser induced chlorophyll fluorescence (LICF) detetermined by point monitoring, chl content and net photosynthetic rate
- 57. Image recorded froma typical irrigated cassava plant : (a) a colour photo, (b) monochrome image recorded at 687 nm, (c) monochrome image recorded at 760 nm, and (d) ratio image (F687:F760) produced from (b) and (C), with colour ramp as indicated by the legend 57
- 58. Scatter plot diagramof (a) net photosynthesis rate (Pn) and (b) total leaf chl for different varieties of cassava plants 58
- 59. Scatter plot diagramof the red:far red fluorescence intensity ratio (a) F687:F760 ratio from SICF imaging measurement and (b) F685:F735 ratio from point monitoring of LICF. The cut-off lines for discrimination are drawn at the mean ratio value of the adjoining groups 59
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- 63. Conclusions Validation ofdiagnostic test Integration of methods Sanitary and phyto-sanitary standards Seed certification programs and epidemiological studies intended to monitor the distribution and spread of pathogens Modified PCR is adaptable to any experimental objectives Molecular techniques can reduce the time required for assay and increase the sensitivity of assay, allowing detection of pathogen before symptom expression Biosensor have new analytical tools this form allows for a rapid response, is ideal for the plant disease detection 63
- 64. 64 Fabrication of teststrips on ELISA will be an innovative strategy Affinity sensors , although extensively studied, still remain in the laboratory in many cases Thermography being non-specific and susceptible to ambient environment, therefore are less suitable for on-field crop disease detection It is also promising that the enzymatic and affinity sensors can be fabricated for multiple pathogen detection rather for single pathogens Future Prospects