Biosensors and Bioelectronics 93 (2017) 72–86

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Biosensors and Bioelectronics

journal homepage: www.elsevier.com/locate/bios

Biosensors for plant pathogen detection MARK

a,b a a,c,⁎
Mohga Khater , Alfredo de la Escosura-Muñiz , Arben Merkoçi
a
Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and Barcelona Institute of Science and Technology, Campus UAB, 08193 Barcelona,
Spain
b
On leave from Agricultural Research Center (ARC), Ministry of Agriculture and Land Reclamation, Giza, Egypt
c
ICREA, Pg. Lluís Companys 23, 08010 Barcelona, Spain

A R T I C L E I N F O A BS T RAC T

Keywords: Infectious plant diseases are caused by pathogenic microorganisms such as fungi, bacteria, viruses, viroids,
Plant pathogen phytoplasma and nematodes. Worldwide, plant pathogen infections are among main factors limiting crop
Bacteria productivity and increasing economic losses. Plant pathogen detection is important as first step to manage a
Virus plant disease in greenhouses, field conditions and at the country boarders. Current immunological techniques
Biosensor
used to detect pathogens in plant include enzyme-linked immunosorbent assays (ELISA) and direct tissue blot
Point-of-care
immunoassays (DTBIA). DNA-based techniques such as polymerase chain reaction (PCR), real time PCR (RT-
Nanomaterial
Antigen PCR) and dot blot hybridization have also been proposed for pathogen identification and detection. However
DNA these methodologies are time-consuming and require complex instruments, being not suitable for in-situ
analysis. Consequently, there is strong interest for developing new biosensing systems for early detection of
plant diseases with high sensitivity and specificity at the point-of-care. In this context, we revise here the recent
advancement in the development of advantageous biosensing systems for plant pathogen detection based on
both antibody and DNA receptors. The use of different nanomaterials such as nanochannels and metallic
nanoparticles for the development of innovative and sensitive biosensing systems for the detection of pathogens
(i.e. bacteria and viruses) at the point-of-care is also shown. Plastic and paper-based platforms have been used
for this purpose, offering cheap and easy-to-use really integrated sensing systems for rapid on-site detection.
Beside devices developed at research and development level a brief revision of commercially available kits is also
included in this review.

1. Introduction as a conventional first step for plant disease diagnosis but it fails in
detecting the presence of pathogen in early infection stages when plant
Plant pathogens are one of the causes for low agricultural produc- infections are symptomless..
tivity worldwide. Main reasons are new, old and emerging plant Early detection of plant pathogens plays an important role in plant
infectious diseases. Their rates of spread, incidence and severity have health monitoring. It allows to manage disease infections in green-
become a significant threat to the sustainability of world food supply house systems and in the field during different stages of plant disease
(Pimentel et al., 2005; Oerke, 2006; Roberts et al., 2006; Savary et al., development and also to minimize the risk of the spread of disease
2012). Despite the lack of sufficient information for the economic infections as well as to prevent introduction of new plant diseases,
losses, it was reported from plant disease loss estimates in U.S state of especially quarantine pathogens at country boarder (Anderson et al.,
Georgia that total losses caused by plant diseases and their control 2004; Strange and Scott, 2005; Brassier, 2008; Vincelli and Tisserat,
costs reached roughly 647.2 million dollars in 2006 and then continued 2008; Miller et al., 2009). Many strategies have been widely used for
up to 821.85 million dollars in 2013 (Martinez, 2006, 2013). Top ten diagnosing plant disease problems including DNA-based methods and
list of economically and scientifically important plant pathogens immunoassays, for the detection of pathogen protein and nucleic acid
includes fungi, bacteria and viruses (Dean et al., 2012; Mansfield extracted from infected plant materials, as direct laboratory based
et al., 2012; Scholthof et al., 2011; Rybicki, 2015) (Table 1). techniques in addition to visual inspection of plant symptoms in the
Plants display different symptoms on leaves, stems and fruits due to field (López et al., 2003) (Fig. 2A).
plant disease infections (López et al., 2003; Al-Hiary et al., 2011) On the other hand there are other indirect strategies based on
(Fig. 1). These symptoms are particularly useful for visual observation analysis of volatile organic compounds (VOC) that plants release as

⁎
Corresponding author.
E-mail address: [email protected] (A. Merkoçi).

http://dx.doi.org/10.1016/j.bios.2016.09.091
Received 15 June 2016; Received in revised form 15 September 2016; Accepted 26 September 2016
Available online 28 September 2016
0956-5663/ © 2016 Elsevier B.V. All rights reserved.
M. Khater et al. Biosensors and Bioelectronics 93 (2017) 72–86

Table 1 Weemen and Schuurs, 1971; Garnsey et al., 1993; Cambra et al., 2000;
Top ten important plant pathogenic bacteria, fungi and viruses published by Molecular Nolasco et al., 2002; Holzloehner et al., 2013; Escoffier et al., 2016).
Plant pathology (Dean et al., 2012; Mansfield et al., 2012; Scholthof et al., 2011; Rybicki,
Immunoassay technology using monoclonal antibodies offers a high
2015).
specificity for plant virus detection, being ideal for testing large scale
Plant Fungi Bacteria Virus plant samples and for the on-site detection of plant pathogens, as done
pathogen with tissue print ELISA and LF devices. In contrast, nucleic acid based
methods are more accurate and specific enough to detect single target
1 Magnaporthe oryzae Pseudomonas Tobacco mosaic
syringae virus pathogen within a mixture containing more than one analyte and
2 Botrytis cinerea Ralstonia Tomato spotted highly effective for detection of multiple targets.
solanacearum wilt In spite of these advantages, molecular detection methods have
3 Puccinia spp. Agrobacterium Tomato yellow some limitations in detecting pathogens at low titres in materials such
tumefaciens leaf curl
as seeds and insect vectors or at early infection stages. Furthermore,
4 Fusarium Xanthomonas oryzae Cucumber
graminearum mosaic false negative results can be produced from cross contamination with
5 Fusarium oxysporum Xanthomonas Potato virus Y PCR reagents which completely block amplification of target DNA,
campestris while false positive results can be generated by cross-amplification of
6 Blumeria graminis Xanthomonas Cauliflower
PCR-generated fragments of non-target DNA. Another limitation is
axonopodis mosaic
7 Mycosphaerella Erwinia amylovora African cassava related to the disability to apply PCR for plant pathogen detection in
graminicola mosaic the field (Louws et al., 1999; Schaad and Frederick, 2002; López et al.,
8 Colletotrichum spp Xylella fastidiosa Plum pox 2003; Martinelli et al., 2015). To overcome such limitations, innovative
9 Ustilago maydis Dickeya (dadantii and Brome mosaic and portable biosensors have emerged in the last years, being widely
solani)
used as diagnostic tools in clinical, environmental and food analysis.
10 Melampsora lini Pectobacterium Potato virus X
carotovorum Pathogen biosensing strategies are based on biological recognition
using different receptors such as antibodies, DNA probe, phage and
others (Eggibs, 2002; Sadanandom and Napier, 2010; Singh et al.,
defense mechanism against pathogen attack (Scala et al., 2013) 2013) (Fig. 3).
(Fig. 2B). Some recent reviews have described in detail the strategies Antibody-based biosensors can allow sensitive and rapid qualitative
for monitoring of volatile compounds in plants for disease detection and quantitative analysis of pathogens offering also label-free possibi-
(Sankaran et al., 2010; Nezhad, 2014; Fang and Ramasamy, 2015; lities. It is important to note that this general approach is limited by the
Martinelli et al., 2015). quality of the antibody employed and its storage condition that could
Several previous studies addressed plant disease diagnosis and affect antibody stability. Also pathogen size can interfere in some
pathogen detection using nucleic acid -based methods, mainly consist- measurements such as the ones based on surface plasmon resonance
ing of polymerase chain reaction (PCR) followed by DNA hybridization (SPR). DNA based biosensors show advantages over antibody based
detection, to determine the genetic content of pathogen (Lin et al., ones mostly related to their better sensitivity thanks to the use of
1990; Minsavage et al., 1994; Anwar Haq et al., 2003; Das, 2004; nucleic acid amplification techniques, which allows to detect plant
Teixeira et al., 2005; Li et al., 2006; Lacava et al., 2006; Saponari et al., pathogen before appearance of disease symptoms. However, they have
2008; Urasaki et al., 2008; Fang et al., 2009; Li et al., 2009; Ruiz-Ruiz some limitations related to the selection and synthesis of specific DNA
et al., 2009; Gutiérrez-Aguirre et al., 2009; Yvon et al.,2009). probes as well as to the fact that detecting short DNA sequence of long
Alternatively, immunoassays, also known as serological assays, includ- double stranded DNA is a common problem in applying biosensing
ing enzyme-linked immunosorbent assay (ELISA), lateral flow devices systems for DNA detection (Skottrup et al., 2008; Fang and Ramasamy,
(LF), tissue print ELISA or direct dot blot immunoassay (DTBIA) have 2015; Hushiarian et al., 2015). Recently, phage-based DNA biosensor
been used to detect the pathogen antigens (Avrameas, 1969; Van for sensing and targeting bacterial plant pathogens has been reported

Fig. 1. Illustration of bacterial disease symptoms on citrus leaves and fruits. Adapted with permission from < http://www.crec.ifas.ufl.edu > (Viewed on Sunday, 22, May 2016).

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Fig. 2. (A) Schematic representation of the procedure for leaf extraction for pathogenic protein and DNA detection based on ELISA and PCR respectively. (B) Illustration of the green
leaf volatiles (GLVs: jasmonic acid (JA), salicylic acid (SA) and ethylene (ET)) released during herbivory, pathogen infection and abiotic stress. Adapted with permission from Scala et al.
(2013).

(Fang and Ramasamy, 2015). Bioluminescent-phage based technology 2.1.1. Voltammetric detection based on the use of enzymes
was developed for determination the presence of Pseudomonas can- In the last three decades, enzyme-linked immunosorbent assay
nabina pv alisalensis that infects cruciferous vegetables (Schofield (ELISA) has become the most widely used serological technique in
et al., 2013). The major advantage of this technology is that detecting diagnostics since the first publication on using ELISA to quantify rabbit
nucleic acid of only viable bacterial cells, and as a result, no false IgG levels (Engvall and Perlmann, 1971). Enzyme immunoassay has
positive was obtained. Nevertheless, the reporter phage expression can been coupled with electrochemical detection methods to diagnose both
be inhibited by presence of some chemical compounds in the tested clinical and plant pathogens with higher sensitivity and selectivity
leaves such as thioethers glucosinolate and isothiocyanate. (Rossier and Girault, 2001; Sarkar et al., 2002; Paternolli et al., 2004).
Along the following sections, the most representative examples of This electrochemical enzyme-linked immunoassay (ECEIA) has incor-
antibody-based and DNA-based plant pathogen detection methods porated enzyme catalysis (enzyme label- substrate complex in presence
using optical and electrochemical techniques are summarized (see of H2O2) followed by electrochemical reducing reaction through
Table 2), also discussing advantages and limitations. An overview amperometric and voltammetric techniques (Zhang et al., 1995a,
about the commercially available devices will also be shown together 1995b; Lee et al., 2005). Stable voltammetric peaks are achieved by
with concluding remarks. controlling the pH of both enzymatic reaction and electrolyte solutions.
Highly preferred is the use of Horseradish peroxidase (HRP) and
alkaline phosphatase (AP) as enzyme labels since they have a variety of
2. Antibody-based biosensors suitable substrates to reach the required sensitivity (Thompson et al.,
1991; Jiang et al., 1995). Despite of high sensitivity of these sensing
2.1. Electrochemical immunosensors systems, the low availability of enzyme-conjugated antibodies repre-
sents an important limitation. Furthermore, enzymatic products can be
Most of the reported electrochemical immunosensors for plant highly affected by the pH of the electrolyte solution being another
pathogen detection are based on label-free technologies (impedimetric drawback in case of enzymatic reactions occurring in the same medium
and quartz crystal microbalance-based ones) and enzymatic label- of the final electrochemical measurement.
based voltammetric approaches on mercury, gold and carbon electro- Jiao et al. (2000) applied a voltammetric indirect ELISA based on
des as detailed in the following sections. horseradish peroxidase (HRP) detection system to detect the plant

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Fig. 3. Schematic of pathogen identification strategies using different biological recognition probes including antibodies, DNA probe, phage, PDPs (phage display peptides) and RBPs
(phage receptor binding proteins). Adapted with permission from Singh et al. (2013).

virus called Cucumber mosaic virus (CMV) using two different HRP antigen which is either a purified CMV or leaf extract prepared by
substrates: o-aminophenol (OAP) and o-phenylenediamine (OPD). grinding infected nicotiana leaves with PBS buffer; (ii) incubation with
Such indirect ELISA has three main steps: (i) immobilization of virus specific antibody for CMV detection; (iii) immunoreaction with sec-

Table 2
Summary of various biosensing techniques used for plant pathogen detection.

Biosensors Detection Assay format Sensing plant pathogen Detection limit

Antibody-based Electrochemical/Enzyme label • Voltammetric Enzyme-based detection • Cucumber mosaic virus • 0.5 ng/ml
Electrochemical/AuNPs tag • Pantoea
stewartii
stewartii sbusp. • 7.8×10 cfu/ml
3

Electrochemical/label-free • Electrochemical impedance spectroscopy (EIS)-based • Plum pox virus • 10Notpg/ml

detection • Prunus necrotic ringspot virus • 250 ng/ml.
reported
• Maize chlorotic mottle virus •
• Quartz crystal microbalance-based approaches
Optical/AuNPs tag • Lateral Flow immunoassays • Potato virus x • 210ng/ml
• Pantoea
Stewartii
stewartii sbusp. • cfu/ml
5

Optical/ fluorescent tag • Fluorescent approaches • Pantoea stewartii sbusp. • 106×10cfu/ml

3

Stewartii • 1.0 ng/ml

5
cfu/ml
• Acidovorax avenae subsp. • 20.5 ng/ml
citrulli • 35.3 ng/ml
• Chilli vein-banding mottle virus •
• Watermelon silver mottle virus
• Melon yellow spot virus
Optical/ label free • Surface plasmon resonance (SPR) systems • Cymbidium mosaic virus • 4842 pg/ml
• Odontoglossum ringspot virus • pg/ml
DNA-based Electrochemical/label-free • DNA hybridization voltammetric detection • Plum pox virus • 12.8 pg/ml
• sugarcane white leaf disease • 4.7 ng/μl
• Trichoderma harzianum • 10 mol/L
−19

Optical/AuNPs tags • Lateral Flow immunoassays • Acidovorax avenae subsp. • 0.48 nM

• AuNPs aggregation-based DNA analysis Citrulli • 0.13 nM
• bridging flocculation • Banana bunchy top virus • 15 ng/ml
• Pseudomonas syringae
Optical/magnetic tag • Pseudomonas syringae • 0.5 ng/μl
Optical/fluorescent tag • Fluorescent approach in DNA microarrays • Botrytis cinerea • 150fMfM
Optical/ luminescent tag • Electrochemiluminscence-based DNA detection • Banana streak virus • 50 fM
• Banana bunchy top virus •
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Fig. 4. Example of electrochemical enzyme-linked immunoassay (ECEIA) sensor using gold nanoparticles as carriers of enzyme-labeled antibodies for signal amplification, applied for
Pantoea stewartii sbusp. Stewartii (PSS) plant bacterial pathogen detection, together with the voltammetric signals obtained for PSS concentrations in the range of 2.0×107–
4.0×104 cfu/ml. Adapted with permission from Zhao et al. (2014). (B) Representative scheme of electrochemical spectroscopy impedance (EIS) immunosensor for Prunus necrotic
ringspot virus (PNRV) DNA determination on glassy carbon electrodes together with the EIS spectra obtained for different dilutions of infected leaf extracts ranging: 100–0.01%.
Adapted with permission from Jarocka et al. (2013).

ondary antibody labeled with HRP. The current derived from the 2.1.2. Label-free electrochemical impedance spectroscopy (EIS)-
reduction of the enzymatic product is measured by linear sweep based detection
voltammetry using a hanging mercury electrode. The sensitivity found Over two decades ago, impedimetric based immunosensors were
for the ECEIA detection of CMV is almost four to ten times higher introduced by Newman and Martelet using techniques that involve
than that of the standard spectrophotometric ELISA, reaching electrochemical impedance spectroscopy (EIS) which studies the
detection limits as low as 0.5 ng/ml using OAD as substrate, also electrode-solution interface changes and detects that impedance
exhibiting high selectivity against four different pathogens: Tobacco changes produced by biomolecular interactions including DNA hybri-
mosaic virus (TMV), Potato virus Y (PVY), Southern bean mosaic dization and protein immunocomplex formation (Newman et al., 1986;
virus (SBMV), Tomato aspermy virus (ToAV) and Turnip mosaic virus Bataillard et al., 1988; Katz and Willner, 2003; K’Owino and Sadik,
(TuMV). 2005; Prodromidis, 2007; Daniels and Pourmand, 2007). Although
Recently gold nanoparticles have been used as tags to amplify the impedance biosensing systems are sensitive and can effectively trace
analytical signal and significantly enhance the immunological assay's reactions occurring upon, their selectivity in real complex sample is a
sensitivity. As an example, Zhao et al. (2014) presented for the first key problem limiting commercial applications. Most impedimetric
time an ECEIA using gold nanoparticle tags loaded by antibodies biosensors are in label-free format and use self-assembled monolayers
labeled with HRP to detect Pantoea stewartii sbusp. stewartii (PSS) (SAMs) as immobilization method to obtain well-ordered monolayers
plant bacterial pathogen (Fig. 4A). on the surface of the electrode and achieve better antibody-antigen
Linear voltammetric measurements were done in PBS solution interaction efficiency (Kausaite-Minkstimiene et al., 2010).
containing hydroquinone (HQ) as enzyme substrate and H2O2 as Thiol SAMs formation on gold electrodes is the most reported
oxidant agent for monitoring the reduction of benzoquinone (BQ). In substrate (Porter et al., 1987; Love et al., 2005) that has been used for
comparison to conventional ELISA assay, the ECEIA for PSS detection impedimetric detection of plant pathogens. One of these approaches
was 20 times more sensitive, reaching a detection limit of 7.8×103 cfu/ has been reported by Jarocka et al. (2011) for Plum pox virus (PPV)
ml. Besides sensitivity, this approach enabled sensitive and specific detection on gold electrodes, taking also advantage of AuNPs for stable
detection of the PSS antigen against other plant bacterial diseases such antibody immobilization while retaining higher biological activity.
as panicle blight, leaf streak and Cercospora leaf spot on rice together Anti-PPV antibodies immobilized onto a 1,6-hexanedithiol/AuNPs
with black spot of crucifer. modified gold electrode were used for the recognition of purified

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PPV. Leaf extract from infected leaves of nicotiana was also prepared receptor for the analyte), detection pad, made of nitrocellulose where
and used as plant virus antigen sources for analysis. The resulting test line (TL) and control line (CL) are printed and adsorption pad, also
impedimetric PVV immunosensor was more sensitive than conven- made of cellulose (Quesada-González and Merkoçi, 2015). Sandwich
tional detection methods assayed, such as AgriStrip rapid immuno- and competitive lateral flow immunoassays (LFIAs) are the main LF
chromatographic assay, being able to detect the presence of 0.01% of formats. In a typical sandwich assay, when the sample is added on the
infected plant material in the diluted healthy samples with a detection sample pad the liquid starts flowing to the conjugate pad where the
limit of 10 pg/ml. analyte (if present) is linked to the label particles, previously con-
The same group later reported a similar approach for the detection jugated with a specific bioreceptor. The conjugate flows by capillarity
of Prunus necrotic ringspot virus (PNRSV) using in this case glassy along the detection pad to the absorbent pad, passing through the TL,
carbon electrodes as platforms and transducers (Jarocka et al., 2013). where it is captured only if the sample has the analyte (positive
In this case, they took advantage of protein A, covalently connected to response), and to the CL, being here always captured, evidencing that
the electrode, for anti-PNRSV antibodies immobilization (Fig. 4B). The the assay works.
as-prepared immunosensor was incubated for 30 min with leaf extracts In addition to antibodies, aptamers and DNA probes are employed
from healthy and PNRSV infected cucumber leaves. The stepwise as biological recognition elements which can be labeled with AuNPs,
preparation of the immunosensor was verified with electrochemical magnetic nanoparticles, fluorescent nanoparticles and enzymes among
impedance spectroscopy (EIS) and cyclic voltammetry (CV) observing others so as to generate the color evolution at the test line. The
the expected increase in the electron-transfer resistance (Rct), which advantages of LFIAs in terms of rapidity, stability and direct on-site
was directly measured with EIS. The immunosensor displayed a very analysis make them one of the most popular diagnostic tools in medical
good sensitivity and selectivity against Plum pox virus (PPV) and was diagnostics, food safety, environmental analysis and plant disease
able to detect PNRSV in plant materials diluted up to ten thousand- detection. Workings with LFIAs have demonstrated very interesting
fold. opportunities for signal enhancements via use of nanomaterials
(nanoparticles, graphene etc.) in addition to simple changes in plat-
2.1.3. Label-free quartz crystal microbalance-based approaches form architecture including vertical flow format. (Parolo et al., 2013a,
Quartz crystal microbalance (QCM) biosensors are based on 2013b; Rivas et al., 2014, 2015; Morales-Narváez et al., 2015; Nunes-
recording changes in oscillation frequency on the surface of the crystal Pauli et al., 2015). The first LFIA for plant pathogen detection was
that produce electrical field (Kanazawa and Gordon, 1985). QCM-based designed to detect Tobacco mosaic virus (TMV) (Tsuda et al., 1992).
immunosensors are highly sensitive and allow label-free detection.. Since this first design, LFIAs have been proposed for the detection of
Many applications have been reported for detecting foodborne patho- several plant pathogens (Danks and Barker, 2000). Particularly, LFIAs
gens as well as on environmental and clinical analysis (O'sullivan and in sandwich format using AuNPs tags have been utilized to plant
Guilbault, 1999; Si et al., 2001; Pohanka et al., 2007; Liu et al., 2007; viruses such as Citrus tristeza virus (CTV) and Potato virus X (PVX)
Bragazzi et al., 2015). Since their use in identifying orchid viruses as a and also plant pathogenic bacteria like Erwinia amylovora, Banana
first application for plant disease detection (Eun et al., 2002), a number xanthomonas and Pantoea stewartii as will be commented in the
of piezoelectric label-free immunosensors based on the use of QCM for following paragraphs.
plant disease determination has been rightly reviewed by Skottrup et al. Salomone et al. (2004) developed a LFIA using standard antibody-
(2008), and continued in the last years presenting multiplexed detec- based sandwich format and AuNPs as label to detect Citrus tristeza
tion of three significant plant pathogenic bacteria (Papadakis et al., virus (CTV) from citrus leaves and fruits. Qualitative results showed
2015). We highlight here the recent approach reported by Huang et al. sensitivity as high as ELISA test with good correlation. The specificity
(2014) developing QCM immunosensor based on self-assembled of the assay was also acceptable, obtaining a level of 5% of false positive
monolayers (SAMs) for identification of Maize chlorotic mottle virus results.
(MCMV). SAMs were formed on the gold surface of QCM crystal layer A similar approach was later developed for the identification of
by layer using mercaptopropanoic and mercaptoundecanoic acids and Potato virus x (Drygin et al., 2012). The reported sensitivity was found
antibodies specific to MCMV. Quantification measurements were to reach 2 ng/ml while selectivity was tested against major potato seed
obtained by observing the changes in the QCM crystal frequency. viruses such as Potato virus Y (PVY), Potato virus M (PVM) and Potato
This biosensor showed a similar sensitivity as ELISA, recording limit of virus A (PVA). Very recently, Feng et al. (2015) performed a rapid
detection of 250 ng/ml. Moreover the developed immunosensor detection of Pantoea stewartii sbusp. stewartii (PSS) extracted from
showed high selectivity against similar viruses, such as Maize Dwarf corn seed samples using a LFIA (Fig. 5A). The LFIA was performed in
Mosaic Virus (MDMV) and Wheat streak mosaic virus (WSMV). the presence of three other plant pathogenic bacteria (Burkholderia
In spite of their high sensitivity, QCM-based measurements are glumae, Xanthomonas oryzae and Pseudomonas syringae) and none
highly affected by the environmental conditions, representing an were detected, evidencing an excellent selectivity. The assay displayed a
important limitation that should be solved for point-of-care applica- detection limit of 105 cfu/ml of PSS..
tions. In addition to the use of AuNPs for colorimetric detection,
fluorescent tags have also been proposed in LFIAs for plant pathogen
2.2. Optical immunosensors detection. This is the case of lanthanide chelate-loaded silica nanopar-
ticles that were used for the determination of Pantoea stewartii sbusp.
Main optical immunosensors for plant pathogen detection are stewartii (PSS), the bacterial pathogen of Stewart's wilt in sweet corn
based on lateral flow devices (paper-based sensors), fluorescence (Zhang et al., 2014). Samples from healthy and infected corn seeds
approaches and surface plasmon resonance (SPR) systems as explained were analyzed following the standard sandwich assay format. The
in the following sections. fluorescence strips allowed to detect a low concentration of PSS
(103 cfu/ml) in less than 30 min with limit of detection hundredfold
2.2.1. Lateral flow immunoassays (LFIAs) lower than ELISA and AuNPs labeled strips.
Paper-based sensors are well known advantageous devices for In spite of their great advantages, LFIAs suffer important limita-
diagnostics applications (Parolo and Merkoçi, 2013). Lateral flow tions related to their low sensitivity and only qualitative/semiquanti-
(LF) is a paper analytical device, also known as immunochromoto- tative results. Although the sensitivity is highly improved using
graphic strip, composed of four different pads: sample pad, made of fluorescent tags as alternative to traditional colorimetric ones, the
cellulose, where the sample is dropped; conjugate pad made of glass need of fluorescence reader (no visual detection possibilities) is an
fiber, impregnated with the bioconjugates solution (label particle and a important limitation for rapid and in-field qualitative analysis. For this

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Fig. 5. (A) Scheme of a lateral flow immunoassay (LFIA) based on gold nanoparticles designed for detection of Pantoea stewartii sbusp. Stewartii (PSS) plant pathogenic bacteria and
pictures of the strips for pathogen concentrations from 1×107 to 1×105 cfu/ml. Adapted with permission from Feng et al. (2015). (B) Scheme of magnetic microsphere immunoassay for
multidetection of Watermelon silver mottle virus (WSMoV) and Melon yellow spot virus (MYSV) plant viruses. Adapted with permission from Charlermroj et al. (2013).

reason, colorimetric LFIAs, mainly based on AuNPs, are still the most pathogenic microorganism causing microbial contamination, food
commonly used for point-of-care analysis. spoilage and plant infection (Vanregenmortel and Pellequer, 1993;
Deisingh and Thompson, 2004; Bergwerff and Van Knapen, 2006;
2.2.2. Fluorescent approaches Mazumdar et al., 2008; Dudak and Boyacı, 2009). The most important
Microsphere sandwich immunoassay technology based on fluores- advantages of this technique rely on the label-free possibilities together
cence-loaded magnetic microsphere and fluorophore- antibodies has with their ability to effectively measure/follow the bioaffinity reactions.
been applied for detecting multiple analytes such as biomarkers, food Since more than two decades ago, the first SPR biosensor for
and plant pathogens (Bergervoet et al., 2008; Kim et al., 2010; Tobacco mosaic virus (TMV) was described (Altschuh et al., 1992).
Mushaben et al., 2013). A recent study has taken a direct application SPR based biosensors have been object of a review (Skottrup et al.,
in the use of microsphere immunoassay technology for multiplex plant 2008) so we detail here some representative examples. For instance,
pathogens simultaneously (Charlermroj et al., 2013). Specific antibo- label free biosensors based on SPR were developed to detect plant
dies to plant bacterial pathogen Acidovorax avenae subsp. citrulli pathogens including Cowpea mosaic virus, Tobacco mosaic virus and
(AAC) and three other plant viruses such as Chilli vein-banding mottle Lettuce mosaic virus as plant viruses and Fusarium culmorum,
virus (CVbMV), Watermelon silver mottle virus (WSMoV) and Melon Phyththora infestans and Puccinia striiformis as fungal plant patho-
yellow spot virus (MYSV) were loaded onto a set of fluorescence-coded gens (Boltovets et al., 2002; Zezza et al., 2006; Torrance et al., 2006;
MagPlex microsphere (Fig. 5B). This technology based on measuring Skottrup et al., 2007a, 2007b). A number of different SPR biosenosrs
the fluorescence intensity of R-phycoerythrin tag enables to determine using DNA probe, antibody and aptamer are reported in the literature
the antigen of the four plant pathogens. The limits of detection for AAC, for monitoring plant pathogens (Wang et al., 2004; Candresse et al.,
CVbMV, WSMoV and MYSV are 6×105 cfu/ml, 1.0 ng/ml, 20.5 ng/ml 2007; Lautner et al., 2010). In very recent years, Lin et al. (2014)
and 35.3 ng/ml, respectively. developed a label free SPR immunosensor using gold nanorods
In spite of great advantages, mainly related to sensitivity and ability (AuNRs) for investigation of two viruses of orchid Cymbidium mosaic
to detect multiple pathogens in a single assay, the main limitations of virus (CymMV) or Odontoglossum ringspot virus (ORSV). Antibodies
these systems are related to the complexity of the assay together with specific to orchid viruses were modified with AuNRs as sensing layers
the need of fluorescent readers. that offer a wide spectral region help in decreasing the color inter-
ference problem caused by sample matrix. This technology was
2.2.3. Surface plasmon resonance (SPR) systems exploited for achieving 48 and 42 pg/ml as detection limits for
Surface Plasmon resonance (SPR) technology is based on monitor- CymMV and ORSV, respectively. The stability of the established SPR
ing changes of refractive index on the sensor surface after ligand- biosensing system was not reported while the specificity was investi-
biomolecule interaction. SPR biosensors have been used in detecting gated using mixture of target and non-target viral antigen; perfor-

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mances were compared by observing signal changes due to viral was investigated in this case by differential pulse voltammetry (DPV).
antigen- antibody binding on the surface of AuNRs. The fabricated DNA biosensor detected crude DNA taken from real
In spite of the above mentioned advantages, a serious drawback in samples (fungal mycelia) with high reproducibility, obtaining a detec-
the use of this technology are the non-specific adsorptions onto the tion limit of 10−19 mol/L. High selectivity for identification of
sensor surface that must be carefully controlled. Trichoderma harzianum against other Tricoderma species, probably
mainly due to the high specificity of the designed probe DNA used as
3. DNA-based biosensors bioreceptor, has also been reported.
As final remark we can state that, although label-free DNA
3.1. Electrochemical DNA biosensors hybridization using voltammetric systems (and hybridization indica-
tors) have great advantages in terms of low cost of analysis, not
The majority of electrochemical DNA biosensors for plant pathogen necessity of labeling step and possibility of analysis of small volumes,
determination are based on label-based and label-free voltammetric their poor sensitivity in complex real samples should be carefully
detection of DNA hybridization. Emerging approaches based on DNA considered before their application for plant pathogen analysis.
translocation through nanopores, even though not reported yet for
plant pathogen detection, show enormous potential in this field so they 3.1.2. Nanochannels as emerging tools for electrochemical DNA
are also commented in the following sections. analysis
Nanopore/nanochannel-based technologies are currently one of the
3.1.1. Label-free DNA hybridization voltammetric detection most promising ones for rapid and efficient DNA analysis (De la
DNA hybridization can be monitored in label-free approaches based Escosura-Muñiz and Merkoçi, 2012). Even though no examples of
on amperometric, impedimetric and voltammetric detection including application for plant pathogen determination using this technology are
square-wave voltammetry (SWV), cyclic voltammetry (CV) and differ- found yet in the bibliography, we preview that its enormous potential
ential pulse voltammetry (DPV) (Liu and Tan, 1999; Azek et al., 2000; will make it possible in a short time and thus consider of great
Wang et al., 2003; Gao et al., 2007; Lillis et al., 2007). relevance to include some remarks in this review.
An example of voltammetric approach has been recently reported Nanopore/nanochannel biosensing systems are inspired by the
by Malecka et al. (2014) for the label-free detection of picomolar microparticle counter device patented by Wallace Coulter more than
concentrations of nucleic acid from Plum pox virus (PPV) glassy 60 years ago (Coulter, 1953). It consists in simply measuring changes
carbon electrodes. (Fig. 6A). Detection of the pathogen-related DNA, in the electrical conductance (electric current or voltage pulse) between
with the complementary target immobilized on the electrode was two chambers separated by a microchannel when a micro-sized analyte
monitored by Osteryoung square wave voltammetry (OSWV). passes through it, giving information about mobility, surface charge,
Voltammetric measurement of electron transfer changes due to the and concentration of the analyte. This sensing principle has been
hybridization reaction allowed detecting 22-mer and 42-mer comple- extended in the last decades for nano-sized analytes evaluation using in
mentary target DNA sequences of PPV at 10–50 pg/ml concentration this case nanometric channels, being the ssDNA analysis extensively
range. A good discrimination between infected and healthy leaf reported. The typical approach consists in the monitoring of ssDNA
samples is reported with a detection limit of 12.8 pg/ml but selectivity molecules translocation (electrophoretically driven) through a single
of this technique was not characterized by other phytopathogens.. nanopore (biological or synthetic) which separates two chambers filled
Well-known approaches based on the use of methylene blue (MB) with an electrolyte solution (Kasianowicz et al., 1996; Bayley and
as hybridization indicator have also been recently reported for sugar- Cremer, 2001; Siwy and Howorka, 2010). Such translocation produces
cane white leaf disease (SWLD) detection (Wongkaew and Poosittisak, changes in the constant current measured between the chambers, being
2014). Voltammetric determination of the plant phytoplasma (causal the current pulse length characteristic of each of the 4 DNA bases (A, T,
agent of.SWLD) was carried out at glassy carbon electrode modified C, G). (Fig. 6B). This ability has opened exciting perspectives for DNA
with chitosan. Electrostatic attraction of negatively charged DNA probe sequencing as alternative to conventional real-time PCR. Not only
to glassy carbon electrode coated with cationic chitosan film for more single nanochannels, but also nanochannel arrays have been proposed
efficient DNA immobilization, as alternative to covalent immobiliza- for the electrical detection of DNA hybridization at the point-of-care
tion, were used in most of the previously mentioned approaches. (De la Escosura-Muñiz and Merkoçi, 2010).
Electrochemical detection of hybridization between ssDNA probe and In addition to DNA, other molecules such as proteins or toxins have
target was performed by cyclic voltammetry (CV) and differential pulse been detected using the single-nanochannel technology (De la
voltammetry (DPV), using MB as a redox indicator, which is covalently Escosura-Muñiz and Merkoçi, 2016). We would like also to highlight
attached to guanine bases. The electrochemical reduction of MB here very recent approaches reported on filamentous virus transloca-
decreased after DNA hybridization due to unavailable guanine bases tion monitoring through silicon nitride membranes (McMullen et al.,
in dsDNA as a complete form, as expected. Following this strategy, a 2014) (Fig. 6C). The size and shape of this kind of virus is ideal for the
detection limit of 4.7 ng/μl of SWLD DNA was obtained, also distin- analysis using these systems, since their stiffness avoid hernias
guishing between target DNA from diseased sugarcane and non-target formation making them able to pass through the channels in elongated
DNA from both healthy and infected sugarcane plants with other forms, and generating well resolved signatures (easy distinguishable of
pathogens like Sugarcane mosaic virus. This biosensor showed good the ones coming from virus collisions with the membrane), opening the
stability of DNA probe immobilization onto the chitosan with interest way to reliable future label-free virus detection systems. Such systems
to develop an effective specific DNA biosensor. could be applied for filamentous virus affecting plants from different
Gold electrodes modified with nanocomposite membranes made of genus like closterovirus (CTV) and potyvirus (PVX).
chitosan (CHIT) and zinc oxide nanoparticles (ZnO NPs) were also
proposed as platforms for developing sensors for plant pathogen DNA 3.2. Optical DNA sensors
voltammetric detection based on MB redox indicator. This kind of
composite can improve the efficiency of the DNA probe immobilization Colorimetric detections of gold nanoparticles (AuNPs) in both
thanks to its good biocompatibility and an enhanced electrochemical lateral flow assays and in aggregation tests, together with the use of
conductivity. Such is the case of the system recently proposed for fluorescent and colorimetric based microarrays and electrochemiluni-
Trichoderma (soil born fungi) determination (Siddiquee et al., 2014). nescence analysis are the most representative examples of optical
Hybridization between DNA target of Trichoderma and its comple- approaches for plant pathogen DNA detection. The great potential of
mentary probe immobilized on the nanocomposite modified electrode nanochannel arrays for this purpose is also highlighted in this section.

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M. Khater et al. Biosensors and Bioelectronics 93 (2017) 72–86

Fig. 6. (A) Example of label-free DNA hybridization approach based on voltammetric analysis applied for Plum pox virus (PPV) detection. Voltammetric signals correspond to 1–8 pM.
Adapted with permission from Malecka et al. (2014). (B) Scheme of the ssDNA translocation through a single α-hemolysin pore and the associated electrical signatures as potential tool
for pathogen DNA sequencing: each of the four bases produces characteristic time series recordings. Adapted from Kasianowicz et al. (1996) with permission. (C) Illustration of a
filamentous virus translocation through a single nanopore drilled on silicon nitride membranes (up) and its signatures discriminated from collisions (down) Adapted from McMullen
et al. (2014) with permission.

3.2.1. Lateral flow assays based on AuNPs monitored and a linear calibration plot was found between peak area of
DNA detection on lateral flow (LF) test strips have been developed test line and different concentrations of target DNA, achieving a
for the analysis of different plant diseases, in most cases using gold detection limit of 0.13 nM. BBTV-DNA lateral flow biosensor achieved
nanoparticle (AuNP)- labeled DNA probes. As example, a competitive higher sensitivity by ten times in comparison to that of electrophoresis.
DNA hybridization format was presented by Zhao et al. (2011) for Selectivity of the strip was evaluated, using plant samples infected with
Acidovorax avenae subsp. Citrulli (AAC) bacterial disease of melons.. other viruses such as Banana streak virus (BSV) and Cucumber
The developed strip allowed reaching a low detection limit of 0.48 nM. mosaic virus (CMV).
The selectivity of the strip was tested against five other plant bacterial
pathogens Xanthomonas campestris, Acidovorax avenae, Clavibacter 3.2.2. AuNPs aggregation-based DNA analysis and related
michiganensis, Pesudomonas. syringae and Erwinia carotovora and approaches
no cross reactivity was observed. AuNPs aggregation-based tests have been extensively used for
Another application of DNA hybridization on lateral flow using biomolecules detection and also recently proposed for the detection
AuNPs-DNA probe was introduced by Wei et al. (2014) for early of plant pathogen DNA. These simple approaches are gaining a great
detection of Banana bunchy top virus (BBTV). In this case, a direct attention for diagnostic applications due to visual detection possibility
sandwich assay consisting in AuNPs-DNA as detection probe and and low cost of analysis.. This is the case of the approach recently
biotinylated-DNA as capture probe was developed. (Fig. 7). reported by Vaseghi and co-workers (Vaseghi et al., 2013) who applied
Qualitative and quantitative measurements of test line color were this principle for Pseudomonas syringae detection (Fig. 8A). The

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M. Khater et al. Biosensors and Bioelectronics 93 (2017) 72–86

Fig. 7. Lateral flow based on DNA hybridization using DNA probe labeled with AuNPs for Banana bunchy top virus (BBTV) detection, qualitative and semi quantitative measurements
using different concentrations of BBTV (from 8×106 to 8×10 copy/μl) nucleic acid (right up) and a bar chart demonstrating its corresponding peak areas of the test line (right down).
Adapted with permission from Wei et al. (2015).

system was tested using other plant pathogenic bacteria such as Huang et al., 2010; Wang et al., 2011). First example of the application
Pseudomonas viridiflava, Pectobacterium cartovortum sub cartovor- of a microfluidic system was described for phytopathology detection
um, Pseudomonas fluoresce, Xanthomonas alfalfae subsp. citrumelo- (Julich et al., 2011). Later a similar approach using microfluidic based
nis, Xanthomonas axonopodis pv. citr and Pseudomonas argenus. The on silver nanoparticle that serves as detection agent (label) enables
results of this assay showed high specificity and sensitivity in detecting visual detection of fungal pathogens of phytophthora species
as low as 15 ng/ml of target DNA of P.syringae. (Schwenkbier et al., 2015). Besides colorimetric approaches, turbid-
Besides gold aggregation mechanism, bridging flocculation is very ity-based microfluidic system was developed for determination of viral
well-known approach in colloid chemistry since its introduction in pathogens infecting orchids such as Cymbidium mosaic virus
1950s (Ruehrwein and Ward, 1952). This kind of approach based on (CymMV) and Capsicum chlorosis virus (CaCV) (Chang et al., 2013;
reversible adsorption to differentiate between long and short DNA Lin et al., 2015).
polymers and has been reported recently for rapid detection significant DNA microarray technology for large scale investigation of gene
plant pathogens (Wee et al., 2015). This method has been applied for expression variations has been developed (Schena et al., 1995) whereas
the visual detection of Pseudomonas syringae as plant bacterial it is difficult to be suited to automation due to the need of several
pathogen and two other devastating pathogenic plant fungi, manual manipulation steps. In 2000s, applications of DNA microarray
Fusarium oxysporum and Botrytis cinerea (Fig. 8B). Key advantage were reported on identification of pathogens causing plant diseases
of flocculation is the reliable detection of the presence of pathogens in (Bonants et al., 2002; Bystricka et al., 2002; Nicolaisen, 2002; Perez-
plants within very early disease stage nevertheless the plants are Ortin, 2002; Sip, 2002; Schoen et al., 2002, 2003; Boonham et al.,
symptomless. Qualitative analysis enabled detecting of isothermal 2003; Mumford et al., 2006; Zhang et al., 2013). Recently Wang and Li
DNA amplicons as little as 0.5 ng/μl. (2007) designed microarray based on DNA sequences labeled with
In spite of the great perspectives of these systems, important fluorescent tags for visual determination of three fungal plant patho-
parameters like the interparticle gap during DNA duplex formation gens (Botrytis cinerea, Didymella bryoniae, and Botrytis squamosa).
should be carefully considered in the design of the assay so as to avoid Glass chip and poldimethylsiloxane (PDMS) have been utilized as
losses in sensitivity. substrates for the developed microfluidic microarray. Fluorescence
signals to the concentrations of target DNA were measured, detecting
3.2.3. Fluorescent and colorimetric approaches in microfluidics and as low as 1 fM of DNA. Despite limited application of microarray on
microarrays systems plant disease detection, microarray allowed flexible DNA probe forma-
Over the last decade, microfluidic chips have been developed as tion, rapid DNA hybridization using small sample volume. A complete
revolution in on-site microbial detection of viruses and bacteria that review addressing microarray application in detecting plant viruses was
infect animals and humans (Figeys and Pinto, 2000; Kricka, 2001; reported (Boonham et al., 2007; Fig. 9A).

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M. Khater et al. Biosensors and Bioelectronics 93 (2017) 72–86

Fig. 8. (A) AuNPs aggregation for detection of DNA of Pesudomonas syringae, an important plant pathogenic bacterium with wide host plant range. Adapted with permission from
Vaseghi et al. (2013). (B) Scheme of a qualitative assay based on bridging flocculation of isothermally DNA amplicons (up) and its application in detecting phytopathogenic fungi
Fusarium f.sp.conglutinans at different infection stages (down). Adapted with permission from Wee et al. (2015).

3.2.4. Electrochemiluminscence-based DNA detection selectivity of ECL assay but the results showed many advantages over
The first report of DNA biosensor using Ru(bpy)3+2 electrochemi- other detection assays including high sensitivity and stability for plant
luminescence (ECL) detection protocol appeared in 1991, in which an virus detection.
excited state emitting light was formed as a result of generation of
electron transfer reaction between two charged species such as Ru and 3.2.5. Nanochannel arrays as emerging platforms for DNA analysis
TPA on electrode surface (Blackburn et al., 1991; Richter, 2004). ECL In addition to their properties for electrochemical analysis, nano-
has various analytical applications in medical diagnosis and environ- porous membranes are also excellent platforms for the development of
mental analysis (Van Ingen et al., 1998). In spite of the excellent optical biosensors for DNA analysis. Some nanoporous materials (i.e.
sensitivity of these systems an important limitation appears within nanoporous alumina and nanoporous silicon) possess optical proper-
solution-based formats that require continuous supply of luminescence ties that are altered by the presence of analytes captured in the inner
reagent. In the recent years, the application of ECL for detection of PCR walls of the nanochannels without the need of any label. Furthermore,
products is described to quantify plant virus nucleic acid. Tang et al. fluorescent tags have also been used for DNA monitoring in nanochan-
(2007) introduced for the first time an improved ECL-PCR detection as nels (De la Escosura-Muñiz and Merkoçi, 2012). As in the case of the
a diagnostic assay in plant virology, taking advantage of magnetic electrochemical ones, these optical approaches have not yet been
beads as a separation tool for the hybridization product exploiting the applied for plant pathogen detection but we consider of great interest
high affinity of biotin-streptavidin. Three plant viruses such as Banana to show here their outstanding potential. The work by Meller’s group
streak virus, Banana bunchy top virus, and Papaya leaf curl virus (McNally et al., 2010) for the optical single-molecule DNA sequencing
were amplified by PCR, then hybridized with a tris(bipyridine) can be selected as illustrative example. A multicolor readout is used
ruthenium (TBR)-labeled detector probe and a capture probe labeled after conversion of the target DNA into a binary code, consisting in the
with biotin. The hybridization products were captured onto streptavi- biochemical conversion of the nucleotides to known oligonucleotides.
din coated magnetic beads and the ECL signal of Ru (bpy)32+ (TBR Hybridization with molecular beacons, using two different fluorophores
label) was generated by using tripropylamine (TPrA) as the co-reactant. was finally detected by translocating the DNA/beacon complex through
This improved ECL-PCR method held a low detection limit down to 50 the nanochannel. Taking advantage of the nanochannels array, the
fM of PCR products through stable ECL signals. Not evaluated is the specific location of each channel in the visual field of the optical

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M. Khater et al. Biosensors and Bioelectronics 93 (2017) 72–86

Fig. 9. (A) Scheme of DNA microarray technique based on DNA hybridization for pathogen characterization applied for Broad bean wilt virus analysis. Red fluorescent pattern
indicates to the presence of virus RNA. Adapted with permission from Boonham et al. (2007), Nezhad (2014). (B) Illustration of the promising optical platforms based on nanochannel
arrays for optical detection of DNA. Adapted with permission from McNally et al. (2010).

detector, allowed the simultaneous readout of the array (Fig. 9B), management and also could reduce the damage caused by plant
which open the way to further applications for multidetection of DNA diseases worldwide. Conventional diagnostic techniques could be time
related to plant pathogens. consuming, are related to special equipment and require still user/
professionals with certain experience. To overcome these difficulties,
4. Commercial available devices recent advances in micro and nanotechnologies have enabled for
developing biosensors for determination of pathogen infections in
Commercial availability of biological recognition elements (i.e. plants using antibody and DNA as biosensing receptors. This work
antibody, DNA probe, aptamer) is a key feature required for successful intensively reviewed the developed antibody-based and nucleic acid-
plant disease diagnosis. To date, most commercialized devices for plant based biosensors in laboratories worldwide for plant disease detection.
pathogen detection based on immunoassays include lateral flow Most DNA biosensors techniques are based on determination of DNA
devices, tissue-print ELISA and plate-ELISA kit (Fig. 10A–D). A variety hybridization events including electroluminescence, fluorescent and
of commercial kits based on immunoassay have been reported in colorimetric approaches in addition to label-free voltammetric etc. In
literature such as pocket kit for orchid virus detection, Agritest lateral spite of advantages of DNA biosensors in terms of sensitivity, selectivity
flow to detect Erwinia amylovora bacterial causal agent of pome trees due to great recognition properties, their in-field application is still
moreover Foresite diagnostic commercial kit for xanthomonas wilt of suffering from sample treatment requisites (eg. DNA extraction). On
banana plant (Braun-Kiewnick et al., 2011; Hodgetts et al., 2015). the other hand, antibody-based scenarios have been developed using
Polyclonal and monoclonal antisera are available on the market for QCM, SPR, fluorescent, voltammetric and label-free impedance detec-
diagnosis of viral, bacterial and fungal disease in plants for commercial tion techniques. Although one would take advantage of high affinity
use. Additionally, DNA & RNA extraction kits have been designed to between antibody and specific antigen (related to plant disease)
isolate total nucleic acid from a variety of plant materials, including uncontrolled antibody immobilization could obstruct reaching efficient
leaves, bark and fruits. Examples of well tested kits are DNeasy and biosensing signal while developing the right detection tool. Although
RNeasy Plant System from Qiagen, ISOLATE plant DNA kit from most of reported biosensors for plant disease detection are still for use
Bioline and GenElute plant genomic DNA from Sigma company. at lab level, it is expected that more portable devices will emerge in the
Emerging mobile applications are helpful tools for farmers in remote future being a strong support for an efficient diagnostic. Given the
areas to detect and identify plant diseases. As example, Gene- Z is a spread of plant disease there is a strong need to develop new biosensors
promising plant disease mobile application based on microfluidic that can be used directly in the field by farmers themselves. Selection of
technology; it has applied on quantification of cancer markers. diagnostic route for plant disease detection relies on the event to be
Lately, Gene- Z is ready to be brought to the market for analyzing analyzed mainly involving (i) phytosanitary analysis and plant quar-
plant pathogen in the field and it can be an interesting solution for an antine (ii) routine large scale surveys and disease risk assessment.
effective monitoring / control of plant disease spread. (Fig. 10E). Sanitary status testing requires the most sensitive method to avoid false
positives and discard any pathogen to have pathogen-free mother
5. Conclusion plants for certification programs. In case of quarantine pathogen
monitoring while import/export of plant materials, experts recommend
In this review we show that early detection of old, new and using more than one diagnostic method to reduce the risk of obtaining
emerging infectious plant disease plays critical role in plant disease false (positives or negatives), therefore nucleic acid-based biosensing

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M. Khater et al. Biosensors and Bioelectronics 93 (2017) 72–86

Fig. 10. Commercial devices for the detection of several plant diseases. (A–C) Lateral flow systems commercialized for phytophthora species, Erwinia amylovora and Potato virus Y
detection. Adapted with permission from: (A) LaChandra bioscience pvt. Ltd. All rights reserved. (B) Abingdon Health Ltd., owner of the trademark Pocket Diagnostic®. All rights
reserved. (C) Loewe® Biochemica GmbH. All rights reserved. (D) ELISA kit for the detection of Citrus tristeza virus, Acidovorax avenae ssp. citrulli and Botrytis cinerea. Adapted with
permission from Loewe® Biochemica GmbH. All rights reserved. (E) Hybrid smart phone application and microfluidic to identify plant pathogen in minutes as promising device not yet
applied on plant disease detection. Adapted with permission from < http://www.treehugger.com > (Viewed on Sunday, 22, May 2016)

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