Plant virus diagnosis elisa

Keywords: plant viruses, diagnostics, next-generation sequencing, omics technologies, nanopore sequencing, quasi-species, in-field analysis. Introduction Viruses are the most genetically diverse organisms that cause infections in plants, animals, and humans. Methods for Plant Viral Diagnostic Numerous methods have been developed and commercialized for plant viral diagnostics.

Open in a separate window. Figure 1. Visual Inspections and Indicator Plants Traditionally, visual inspections of infected plants and seeds were the most common method used for plant pathogen detection. Microscopic Methods One of the most classic methods for visualizing viruses in plant tissues is microscopic detection using modern light and high-resolution electron microscopes [ 31 , 32 ]. Serological Methods Serological methods, such as ELISA enzyme-linked immunosorbent assay , which are based on the reliable detection of a protein molecule using polyclonal or monoclonal antibodies, are widely used in plant viral diagnostics.

Table 1 Serological tests based on ELISA and its modified methods used employed in plant viral disease diagnosis for various important crops. Nucleic Acid-Based Methods Nucleic acid-based methods are widely employed in many fields of diagnosis, including clinical, food safety, and environmental analysis. PCR-Based Methods Polymerase chain reaction PCR is one of the most important methods and is considered the gold standard for the molecular detection of various pathogens.

Figure 2. Next-Generation Sequencing omics -Based Methods Before the emergence of next-generation sequencing technologies, first-generation sequencing Sanger sequencing dominated the field of biological research. Analysis Using Second-Generation Sequencing Technologies Second-generation sequencing technologies evolved based on high-throughput sequencing with the fast delivery of results at a reduced cost.

Table 3 Next-generation sequencing second-generation sequencing platforms -based identification of causative agents associated with the viral diseases of various crops.

Analysis Using Third-Generation Sequencing Technologies Second-generation sequencing transformed the field of biological research and is routinely used in environmental studies of microbial diversity, the human-associated microbiome, plant genomics, etc.

Table 4 Next-generation sequencing third-generation: MinION Nanopore -based identification of causative agents associated with the viral diseases of various crops. First report on infection of P. Current Challenges and Future Prospects Nanopore Technology The management of plant viral diseases requires an effective diagnosis method that can generate early results for analyzing and preventing diseases.

Figure 3. Conclusions Monitoring plant health and quickly detecting pathogens are essential to reduce viral disease spread and facilitate effective management practices. Author Contributions Conceptualization, G. Funding B. Institutional Review Board Statement The study did not require any ethical approval. Informed Consent Statement Patient consent was not required in this review. Data Availability Statement All data generated during this study are included in this article.

Conflicts of Interest The authors declare no conflict of interest. References 1. Rojas M. Emerging plant viruses: A diversity of mechanisms and opportunities. In: Roossinck M. Plant Virus Evolution. Maksimov I. Mechanisms of plant tolerance to RNA viruses induced by plant-growth-promoting microorganisms. Plant viruses infecting Solanaceae family members in the cultivated and wild environments: A review. Pandemics of people and plants: Which is the greater threat to food security?

Gallet R. Vector-transmission of plant viruses and constraints imposed by virus-vector interactions. Seo J. Molecular dissection of distinct symptoms induced by tomato chlorosis virus and tomato yellow leaf curl virus based on comparative transcriptome analysis.

Adams I. Next-generation sequencing and metagenomic analysis: A universal diagnostic tool in plant virology. Plant Pathol. Rubio L. Detection of plant viruses and disease management: Relevance of genetic diversity and evolution. Relative incidence, spatial distribution and genetic diversity of cucurbit viruses in eastern Spain. Mechanisms of viral mutation.

Life Sci. Massart S. Current impact and future directions of high throughput sequencing in plant virus diagnostics. Virus Res. Mandal B. Emerging problems of tospoviruses Bunyaviridae and their management in the Indian subcontinent. Plant Dis.

Maree H. Application of HTS for routine plant virus diagnostics: State of the art and challenges. Villamor D. High-throughput sequencing identifies novel viruses in nectarine: Insights to the etiology of stem-pitting disease.

Stobbe A. Plant virus metagenomics: What we know and why we need to know more. Plant Sci. Malmstrom C. The expanding field of plant virus ecology: Historical foundations, knowledge gaps, and research directions.

Khare E. Multifaceted interactions between endophytes and plant: Developments and prospects. Theuns S. Nanopore sequencing as a revolutionary diagnostic tool for porcine viral enteric disease complexes identifies porcine kobuvirus as an important enteric virus. Martinelli F. Advanced methods of plant disease detection.

A review. Barba M. Historical perspective, development and applications of next-generation sequencing in plant virology.

Scholthof K. Top 10 plant viruses in molecular plant pathology. Naidu R. Bos L. Symptoms of Virus Diseases in Plants. Pudoc; Wageningen, The Netherlands: Webster C. Diagnosis of plant viral pathogens. Virus pathogens in Australian vineyards with an emphasis on shiraz disease.

Tolkach V. Virus diseases of vegetable and melon crops in the South of the Russian far East. Russia Ecol.

Yamaguchi A. Increased respiration of leaves bearing necrotic local virus lesions. Senshu H. A dual strategy for the suppression of host antiviral silencing: Two distinct suppressors for viral replication and viral movement encoded by potato virus M.

Balodi R. Plant disease diagnosis: Technological advancements and challenges. Indian Phytopathol. Mutka A. Image-based phenotyping of plant disease symptoms. Baker K. Electron microscopy: Current applications to plant virology. Horne R. Molecular Plant Virology.

The development and application of electron microscopy to the structure of isolated plant viruses. Steere R. Identification of crystalline inclusion bodies extracted intact from plant cells infected with tobacco mosaic virus. Kitajima E. Electron microscopy in plant virology: Past, present and future. Electron microscopy methods for virus diagnosis and high resolution analysis of viruses.

Song G. Monoclonal antibody-based serological assays for detection of Potato virus S in potato plants. Zhejiang Univ. Guo L. Monoclonal antibody-based serological detection of Rice stripe mosaic virus infection in rice plants or Leafhoppers. Klap C. The potential risk of plant-virus disease initiation by infected tomatoes. Zhang M. Visual detection for nucleic acid-based techniques as potential on-site detection methods.

Zechmann B. Rapid diagnosis of plant virus diseases by transmission electron microscopy. Deng J. Investigation on subcellular localization of Rice stripe virus in its vector small brown planthopper by electron microscopy. El-Deeb S. El-Abhar M. Identification, characterization and ultrastructure aspects of Alfalfa mosaic virus infecting potato Solanum tuberosum L.

Analysis of Prune dwarf virus intercellular transport and pathogenesis. Rong W. Molecular and ultrastructural mechanisms underlying yellow dwarf symptom formation in wheat after infection of barley yellow dwarf virus. Bhardwaj A. Applied Plant Virology. Advance methods for the isolation and characterization of plant viruses infecting crops; pp.

Xie Y. Highly sensitive serological methods for detecting tomato yellow leaf curl virus in tomato plants and whiteflies. Kothandaraman S.

Seed-borne nature of a begomovirus, Mung bean yellow mosaic virus in black gram. Gorovits R. The involvement of heat shock proteins in the establishment of Tomato yellow leaf curl virus infection.

Wieczorek P. Development of a new tomato Torrado virus-based vector tagged with GFP for monitoring virus movement in plants. Hull R. Plant Virology. Kang S. Zamora M. Ertel K. Cryo-electron tomography reveals novel features of a viral RNA replication compartment. Clark M. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. Atkinson P. On the origin of dark green tissue in tobacco leaves infected with tobacco mosaic virus.

Rani A. The future of plant pathogen diagnostics in a nursery production system. Abd El-Aziz M. Three modern serological methods to detect plant viruses.

Monoclonal antibody-based serological assays and immunocapture-RT-PCR for detecting Rice dwarf virus in field rice plants and leafhopper vectors. Nascimento A. A simple kit of plate-trapped antigen enzyme-linked immunosorbent assay for identification of plant viruses. Seepiban C. Development and application of triple antibody sandwich enzyme-linked immunosorbent assays for begomovirus detection using monoclonal antibodies against Tomato yellow leaf curl Thailand virus.

Seal S. The prevalence of badnaviruses in West African yams Dioscorea cayenensis-rotundata and evidence of endogenous pararetrovirus sequences in their genomes. Galvino-Costa S. Molecular and serological typing of Potato virus Y isolates from Brazil reveals a diverse set of recombinant strains.

Kutluk Yilmaz N. Distribution of resistance-breaking isolates of beet necrotic yellow vein virus differing in virulence in sugar beet fields in Turkey. Acta Agricult. B Soil Plant Sci. Wisler G. Uehara-Ichiki T. Detection and diagnosis of rice-infecting viruses. Lima J. Volume Moslih Al-Moslih. Volume 1.

InTech; Rijeka, Croatia: Serology applied to plant virology; pp. Boonham N. Byzova N. Development of immunochromatographic test systems for express detection of plant viruses. Niu Y. Monoclonal antibody-based colloid gold immunochromatographic strip for the rapid detection of Tomato zonate spot tospovirus. Bin Y. Development of an immunochromatographic strip test for rapid detection of citrus yellow vein clearing virus.

A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens. Jeong J. A review of detection methods for the plant viruses. Mirmajlessi S. Real-time PCR applied to study on plant pathogens: Potential applications in diagnosis-a review. Nateqi M. Detection and molecular characterization of the Iris severe mosaic virus-Ir isolate from Iran. Kimathi R. First report of Tomato chlorosis virus infecting tomato in Kenya. Kwon J. Reverse transcription polymerase chain reaction-based system for simultaneous detection of multiple lily-infecting viruses.

Kumar R. Mehle N. Ling K. Molecular characterization of two Pepino mosaic virus variants from imported tomato seed reveals high levels of sequence identity between Chilean and US isolates. Virus Genes. Menzel W. First report of tomato brown rugose fruit virus infecting tomato in Germany.

New Dis. First report of tomato brown rugose fruit virus infecting greenhouse tomato in the United States. Kumar S. Cherian S. Characterization of tobacco mosaic virus isolated from tomato in India.

Nagendran K. The occurrence and distribution of major viruses infecting cucurbits in Tamil Nadu state, India. Vinayarani G. Deng D. Detection and differentiation of whitefly-transmitted geminiviruses in plants and vector insects by the polymerase chain reaction with degenerate primers. Molecular detection and characterization of Tomato leaf curl New Delhi virus causing mosaic disease on bitter gourd in Tamil Nadu, India.

Trends Biosci. Hyun J. Development of multiplex PCR for simultaneous detection of citrus viruses and the incidence of citrus viral diseases in late-maturity citrus trees in Jeju Island.

Sinha S. First report of Cucumber mosaic virus associated with yellowing mosaic disease of African basil Ocimum gratissimum in India. Liu H. Majumder S. Simultaneous detection of four garlic viruses by multiplex reverse transcription PCR and their distribution in Indian garlic accessions.

Van Brunschot S. Development of a multiplexed bead-based suspension array for the detection and discrimination of Pospiviroid plant pathogens. Zheng L. A novel pair of universal primers for the detection of potyviruses.

Recent advances on the multiplex molecular detection of plant viruses and viroids. Mulholland V. Plant Pathology. Immunocapture-PCR for plant virus detection; pp. Koolivand D. Vigne E. Comparison of serological and molecular methods with high-throughput sequencing for the detection and quantification of Grapevine Fanleaf Virus in vineyard samples.

Notomi T. Although fluorescence measurement provides sensitive detection of abnormalities in photosynthesis, the practical application of this technique in a field setting is limited [ 51 , 52 , 53 ]. Hyperspectral imaging can be used to obtain useful information about the plant health over a wide range of spectrum between and nm. Hyperspectral imaging is increasingly being used for plant phenotyping and crop disease identification in large scale agriculture. The technique is highly robust and it provides a rapid analysis of the imaging data.

Furthermore, hyperspectral imaging cameras facilitate the data collection in three dimension, with X- and Y- axes for spatial and Z- for spectral, which contributes to more detailed and accurate information about plant health across a large geographic area [ 40 ].

Hyperspectral techniques have been widely used for plant disease detection by measuring the changes in reflectance resulting from the biophysical and biochemical characteristic changes upon infection. 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 [ 54 , 55 , 56 ].

A completely different non-optical indirect method for plant disease detection involves the profiling of the volatile chemical signature of the infected plants.

The pathogen infections of plants could result in the release of specific volatile organic compounds VOCs that are highly indicative of the type of stress experienced by plants [ 57 ]. VOCs are also produced when green leaf plants are damaged pathogenically and mechanically.

For example, green leaf volatiles GLVs such as cis hexenol, cis -hexenyl acetate and hexyl acetate are reported to have been produced during mechanical damage to plant leaves, i. Profiling of such VOC could be used as a means to identify the type and nature of infection and accordingly be used for disease diagnostics and confirmation [ 57 ].

The volatile signature of plants could be analyzed using gas-chromatography GC technique to analyze the presence of the specific VOC that is indicative of a particular disease [ 60 ]. To enhance the performance of compound separation and analysis, the gas chromatography is often combined with mass spectrometry GC-MS to identify unknown compounds in the volatile sample [ 61 , 62 , 63 ]. It also allows the detection of diseases at different stages based on the quantitative information collected from the VOC sample.

Although various methods for plant disease identification have already been developed and some have been implemented, their application is limited due to multiple reasons: they are either time consuming, destructive, demand a skilled technician, require laboratory set-up, do not provide real-time monitoring e.

Growers are interested in a solution that could help them identify pathogen infections in crops in a rapid, real-time and non-destructive fashion so that timely intervention and preventative treatments can be performed to contain the infection and minimize the crop losses.

This would allow the growers to save millions of dollars in fungicide costs, by allowing them to localize sprayings and timely applications rather than preemptive spray massive regions of crop field. A wide variety of sensors have been developed and commercialized for various applications including environmental monitoring and medical diagnostics. Depending on the operating principle of the sensor, the analytes could be detected using a sensor based on electrical, chemical, electrochemical, optical, magnetic or vibrational signals.

The limit of detection could be enhanced by the use of nanomaterial matrices as transducers and the specificity could be enhanced by the use of bio-recognition elements such as DNA, antibody, enzymes etc.

Recent breakthroughs in nanotechnology enable the preparation of various nanoparticles and nanostructures with few technical hurdles. Nanoparticles display fascinating electronic and optical properties and can be synthesized using different types of materials for electronics and sensing applications [ 64 ]. For biosensing application, the limit of detection and the overall performance of a biosensor can be greatly improved by using nanomaterials for their construction.

The popularity of nanomaterials for sensor development could be attributed to the friendly platform it provides for the assembly of bio-recognition element, the high surface area, high electronic conductivity and plasmonic properties of nanomaterials that enhance the limit of detection. Various types of nanostructures have been evaluated as platforms for the immobilization of a bio-recognition element to construct a biosensor.

The immobilization of the biorecognition element, such as DNA, antibody and enzyme, can be achieved using various approaches including biomolecule adsorption, covalent attachment, encapsulation or a sophisticated combination of these methods.

The nanomaterials used for biosensor construction include metal and metal oxide nanoparticles, quantum dots, carbon nanomaterials such as carbon nanotubes and graphene as well as polymeric nanomaterials. Nanoparticles have been utilized with other biological materials such as antibody for detecting Xanthomonas axonopodis that causes bacterial spot disease [ 65 ].

Gold nanoparticle-based optical immunosensors have been developed for detection of karnal bunt disease in wheat using surface plasmon resonance SPR [ 66 ]. In addition to single probe sensors, nano-chips made of microarrays which contain fluorescent oligo probes were also reported for detecting single nucleotide change in the bacteria and viruses with high sensitivity and specificity based on DNA hybridization [ 67 ]. Fluorescent silica nanoparticles FSNPs combined with antibody as a biomarker have been studied as the probe, which successfully detected plant pathogens such as Xanthomonas axonopodis pv.

Vesicatoria that cause bacterial spot diseases in Solanaceae plant [ 65 ]. Quantum dots QD have also been used for biosensor construction for disease detection [ 68 ]. Due to their unique and advantageous optical properties, they have been used for disease detection using fluorescence resonance energy transfer FRET mechanism [ 69 ], which describes energy transfer between two light-reactive molecules.

For example, Rhizomania, which is the most destructive disease in sugar beet, is caused by beet necrotic yellow vein virus BNYVV. In addition to QD-based sensors, the use of other novel materials for sensor fabrication have been explored in order to attain high sensitivity and low limits of detection [ 72 , 73 , 74 ].

Gold nanoparticles are widely used nanomaterials due to their high electroactivity and electronic conductivity for electron transfer [ 75 , 76 ]. Recently, nanomaterial-based electrochemical sensors have been reported for plant disease detection by our group [ 77 ]. The application of gold nanoparticle AuNP modified electrode has been reported by our group for the electrochemical detection of methyl salicylate, a key plant volatile organic compound released by plants during infections Figure 1.

Moreover, in addition to gold nanoparticles, semiconductive metal oxide nanoparticles have also been reported for VOC detection due to its advantages such as low cost, suitability for electron conduction for amperometric signal and the ease at which to obtain a desired size and shape.

Our previous work has demonstrated the application of metal oxide nanoparticles such as SnO 2 and TiO 2 for VOC detection such as p -ethylguaiacol produced by infected strawberry Figure 2 [ 57 ]. The limit of detection achieved was in the nanomolar concentration range [ 57 ].

In addition to the detection of plant VOC which is indicative of a particular disease, nanoparticles can also be used for detection of compounds released by the pathogen itself. Different types of phytopathogens—phytobacteria, viruses and fungi—have been detected through nanoparticle based amperometric biosensors [ 65 , 78 , 79 ].

The responses of current to methyl salicylate and sensitivity are shown in the inset. Figure is adopted from Ref.

Compared to the non-specific nanoparticle-based biosensors, inclusion of a bio-recognition element can greatly increase the specificity of the sensor. Consequently, other types of biosensors have been developed and among them affinity biosensors are popular. In affinity biosensors, the sensing is achieved based on the reaction of the bio-recognition element and the target analyte [ 80 ].

Affinity biosensors can be developed using antibody and DNA as recognition elements. Antibodies are versatile and are suitable for diverse immunosensing applications. Antibody-based biosensor allows rapid and sensitive detection of a range of pathogens especially for foodborne diseases and this technique has already been developed for food safety monitoring. The antibody-based biosensors provide several advantages such as fast detection, improved sensitivity, real-time analysis and potential for quantification.

Antibody-based biosensors hold great value for agricultural plant pathogen detection [ 81 ]. The biosensors enable the pathogen detection in air, water and seeds with different platforms for greenhouses, on-field and postharvest storages of processors and distributors of crops and fruits [ 81 ]. The principle of establishing antibody-based immunosensors lies in the coupling of specific antibody with a transducer, which converts the binding event the specific binding of antibody modified on the biosensor with the antigen, e.

Most antibody-based biosensors use one of the following types of electrochemical transducers: amperometric, potentiometric, impedimetric and conductometric. Amperometric biosensing platforms use electric current signal resulting from the specific binding event [ 82 , 83 ].

Potentiometric biosensors on the other hand convert the biorecognition of an analyte into a voltage signal [ 82 ]. Impedimetric biosensors detect analyte by impedance change upon specific combination of the antibody and analyte. Based on the metabolic redox reactions of microorganisms, impedimetric biosensors are often used for biomass detection by microbial metabolism [ 84 ]. The conductometric biosensors are based on conductometric detection where the biological signal is converted to an electrical signal through a conductive polymer, such as polyacetylene, polypyrrole or polyaniline [ 80 ].

Other types of transducers non-electrochemical for affinity biosensing have been developed and reported including surface plasmon resonance SPR , quartz crystal microbalance QCM and cantilever-based sensors.

SPR-based sensors can measure the change in refractive index due to the attachment of the analyte to the metal surface e. A QCM-based sensor detects a mass variation per unit area of the QCM crystal by measuring the change in frequency of a quartz crystal resonator. The QCM crystal is typically modified with a recognition element e. Similar to QCM-based sensors, cantilever-based sensors measure resonance frequency changes upon combination of the analytes and the sensor surface [ 81 ].

Based on the capability of detecting small analytes such as nucleic acid and proteins, cantilever-based sensors have been used for detection of pathogenic microorganisms [ 87 ]. The change can be translated into a measurable signal for detection. During the past decade, many articles have been published demonstrating the capability of antibody-based biosensors for detection of plant pathogens such as Cowpea mosaic virus , Tobacco mosaic virus , Lettuce mosaic virus , Fusarium culmorum , Puccinia striiformis , Phytophthora infestans , orchid viruses and Aspergillus niger [ 86 , 88 , 89 , 90 , 91 , 92 , 93 , 94 ].

The limit of detection was evaluated to be approximately 1 ppb [ 85 ]. In recent years, antibody-based biosensor technology has seen tremendous progress upon implementation of nanotechnology based approaches for the sensor fabrication.

The QCM technique was able to detect each of the orchid viruses as low as 1 ng [ 86 ]. Other nanomaterials made of polymers such as polypyrrole PPy nanoribbon modified chemiresistive sensors were fabricated by a lithographically patterned nanowire electrode position LPNE technique. The limits of detection of the current antibody based biosensors are proved to be approximately two orders of magnitude higher than that of conventional ELISA methods [ 97 ].

Apart from the common abiotic material for antibody-based biosensor fabrication, biosensors based on living cells are characterized by low limit of detection, high specificity and rapid response time. This study demonstrated an important step towards the development of a portable plant virus detection system suitable for on-field application [ 98 ]. Although the mechanisms, advantages and applications of antibody-based biosensors for plant disease detection have been highlighted, it is important to discuss the limitations.

Since many biosensors based on antibodies focus on specific binding with a particular antigen, issues such as the exposure of a bacterial strain to environmental stress pH and temperature , could cause errors in the measurement. In addition, the immobilization of large bacteria and fungi, whose diameters exceed the SPR range, might compromise the detection.

More importantly, antibodies are vulnerable and are easy to get denatured, which requires specific environment pH, temperature, etc. A recently developed new type of affinity biosensor uses nucleic acid fragments as elements for pathogen detection. The detection of specific DNA sequence is of significance in a variety of applications such as clinical human disease detection, environmental, horticulture and food analysis.

Due to the possibility of detection at a molecular level, the DNA-based biosensor enables early detection of diseases before any visual symptoms appear. The application of specific DNA sequences has been widely used for detection of bacteria, fungi and genetically modified organisms.

Based on the specific nucleic acid hybridization of the immobilized DNA probe on the sensor and the analyte DNA sequence, DNA-based biosensor allows rapid, simple and economical testing of genetic and infectious diseases. There are four major types of DNA-based biosensors depending on their mode of transduction: optical, piezoelectric, strip type and electrochemical DNA biosensors.

Optical DNA biosensors transduce the emission signal of a fluorescent label. The detection of DNA analyte is realized through a variation in physio-chemical properties such as mass, temperature, optical property and electrical property as a result of double-stranded DNA dsDNA hybridization occurs during the analyte recognition Figure 3 b.

Unlike the optical type, piezoelectric DNA biosensors detect the analyte using a quartz crystal that oscillates at a specific frequency at an applied oscillating voltage. Detection of DNA hybridization can also be realized through nanoparticle-based colorimetric detection provided by strip type DNA biosensor. Electrochemical measurements are used for sequence-specific detection of analyte DNA by electrochemical DNA-based biosensors. The current change with constant applied potential can be monitored and used for interpretation of DNA hybridization in amperometric electrochemical DNA biosensor [ 99 ].

Bacterial pathogens are detectable by DNA-based biosensors due to their unique nucleic acid sequence, which can be specifically hybridized with the complementary DNA probe. This is different from the antibody-based biosensors where hydrophobic, ionic and hydrogen bonds play a role in the stabilization of antigen-antibody complex.

Two orchid viruses— Cymbidium mosaic virus CymMV and Odontoglossum ringspot virus ORSV —have been detected with specific oligonucleotide probes with a fluorescent moiety attached to one end of DNA while a quenching moiety attached to the opposite end. Four such molecules have been designed and this technique has been successfully applied to detect viral RNA of both orchid viruses with limits of detection as low as 0.

Although the application of DNA-based biosensors for plant disease detection is promising, PCR may have to be performed prior to the probing process due to the small quantity of nucleic acid present in the bacteria cells [ ]. The fluorescence intensity of all molecular beacons increased significantly and the limit of detection of purified viral RNA was estimated to be 0. Figure has been adopted from Ref. The use of enzyme as bio-recognition element can provide highly selective detection of the target analyte due to the high specificity of enzymes towards the analyte.

An enzyme specific for the analyte of interest is immobilized on the nanomaterial modified-electrode. The amperometric detection is based on the bio-electrocatalytic reaction between the target analyte and electrode, which results in an electrical signal current that can be used for quantitative detection of the analyte. The amperometric signal can be obtained through either direct or mediated electron transfer based electrochemical reactions Figure 6.

Unlike other types of biosensors, which are not widely commercialized, the enzymatic electrochemical biosensors have been successfully commercialized, thanks to the invention of glucose biosensors, which are widely used in personal diabetes monitors [ ].

A similar biosensing methodology can be adopted for plant pathogen detection, food quality detection and environmental monitoring [ ]. For plant pathogen detection, enzymatic biosensors could be used if the target VOC could be collected in the form of a liquid sample. Previous studies have shown that several of phytohormones are catabolized by redox enzymes, offering prospects for using these enzymes for the development of highly selective enzyme-based biosensors for detecting plant chemicals [ ].

Our previous work has already proved the detection of methyl salicylate with a bi-enzymatic system [ ]. Many of the VOCs produced by infected crops are alcohols and aldehydes such as cis hexenol and trans hexanal, which can be catalyzed by alcohol dehydrogenase enzymes. Accordingly, these enzymes can be used for the development of biosensors for the detection of alcohol or aldehyde based VOCs which are specific to the infection. A summary of the different volatiles known to be released due to plant stresses are listed in Table 2 [ 60 ].

In addition to those specific volatile organic compounds, the common phytohormones such as auxin, cytokinins and gibberellins which are indicative of plant health could also be deactivated by oxidases. Gibberellin is deactivated by GAoxidases which provides the potential for fabrication of gibberellin detection for plant disease prediction [ , ]. Although enzyme-based biosensors usually provide high sensitivity and specificity for the detection, stability of enzymes is of major concern.

In addition, the enzyme catalysis varies with factors such as temperature and pH which compromises the accuracy of the biosensor. Schematic illustration of enzymatic biosensor based on A mediated electron transfer and B direct electron transfer DET. VOCs emitted from whole, intact tomato plants or detached leaves, and biotic stress causing agents responsible for increases in VOC emissions [ 60 ]. It infects the bacteria and replicates within the bacteria and finally lyses the bacterial host to propagate.

Being able to lyse the bacteria, bacteriophage has been widely studied and used in phage therapy to cure bacterial infections [ ]. Phage therapy has been used for not only human diseases, but also plant disease control. In addition to phage therapy, bacteriophage is also emerging as a promising alternative for pathogen detection due to its high sensitivity, selectivity, low cost and higher thermostability [ , , ].

Upon the interaction between the bacteriophage and the target analyte, the impedance of charge transfer reactions at the interface changes which is used as a signal for detection. Bacteriophages have demonstrated to be successful in controlling plant pathogens recently such as Dickeya solani , the bacterial infecting of potatoes and tomatoes [ , ].

Tlili et al. Schofield reported a phage-based diagnostic assay for detecting and identifying Pseudomonas cannabina pv. Alisalensis from cultures and diseased plant samples [ ]. The bacterial luxAB reporter genes encoding the luciferase were integrated into P. In the presence of target pathogen cell, the lux AB can be expressed in the host cell due to specific infection through the phage. The expressed luciferase results in the light emission in presence of n-decanal, oxygen and flavin mononucleotide Figure 7 [ ].

In addition to P. Actinidiae , which causes bacterial canker of kiwifruits, and Ralstonia solanacearum , a soilborne bacterium that is the causative agent of bacterial wilt in many important crops [ , , ]. The progress in discovering more bacteriophages provides the possibility for fabrication of more bacteriophage-based sensors for plant disease detection. Schematic illustration of A P. The advantages of using bacteriophage as the recognition element for biosensors are its high selectivity and low cost of the phage.

Furthermore, compared to the antibody-based sensor, bacteriophage-based sensors are more thermostable which allows the detection in different temperature ranges and longer shelf life. PCR can also help farmers detect the presence of pathogens that have long latent periods between infection and symptom development.

Moreover, it can quantify pathogen biomass in host tissue and environmental samples, and at the same time detect fungicide resistance.

PCR-based detection, however, is expensive compared to protein-based diagnostic methods, and also requires costly equipments. So far, PCR kits have been developed to detect black Sigatoka disease in bananas, Phytophthora infestations in potatoes, and Fusarium infection in cotton. This recognition is due to the ability of specific host proteins, called antibodies, to recognize and bind proteins that are unique to a pathogen antigens and to trigger an immune reaction Figure 3a.

Protein-based diagnostic kits for plant diseases contain an antibody the primary antibody that can either recognize a protein from either the pathogen or the diseased plant. Because the antibody-antigen complex cannot be seen by the naked eye, diagnostic kits also contain a secondary antibody, which is joined to an enzyme.

This enzyme will catalyze a chemical reaction that will result in a color change only when the primary antibody is bound to the antigen. The enzyme-linked immunosorbent assay ELISA method makes use of this detection system, and forms the basis of some protein-based diagnostic kits. ELISA kits are very easy to use because test takes only a few minutes to perform, and does not require sophisticated laboratory equipment or training. Some of them detect diseases of root crops e. ELISA techniques can detect ratoon stunting disease of sugarcane, tomato mosaic virus, papaya ringspot virus, banana bract mosaic virus, banana bunchy top virus, watermelon mosaic virus, and rice tungro virus.

It can detect the presence of all races, biovars, and serotypes of Ralstonia solanacearum, the pathogen that causes bacterial wilt or brown rot in potato. With even more advances in molecular biology and immunology, scientists and farmers alike will be able to improve plant disease diagnosis.

Efforts are already underway to produce better diagnostic kits to detect pathogens in crops important to developing countries. Attempts to differentiate closely related strains of APLV or radish mosaic virus by direct ELISA using F ab' 2 fragments either for coating the plates or after labelling with alkaline phosphatase for detecting the trapped antigens failed.

Under suitable conditions, the additional working step usually necessary for indirect ELISA could be avoided by using a short procedure which at low concentrations of detecting antibodies was more sensitive than the conventional procedure.