Current Research

and
Innovations
in
Plant Pathology
Volume - 15

Chief Editor
Dr. Hemant Kumar Singh
Associate Professor,
Department of Plant Pathology, Narendra Deva University of Agriculture
and Technology, Kumarganj, Faizabad, Uttar Pradesh, India

AkiNik Publications
New Delhi
Published By: AkiNik Publications

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Publication Year: 2021
Pages: 129
ISBN: 978-93-91216-89-4
Book DOI: https://doi.org/10.22271/ed.book.1261
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 Contents

Chapters Page No.

1. Host Defence Suppression by Plant Pathogens 01-35
(Meghana Suresh Nayak)

2. Management of Plant Viruses 37-65

(Sompalli Suresh Rao, R. Sarada Jayalakshmi Devi, K.V. Hari Prasad, M.K.
Jyosthna and P. Arunasri)

3. Biosensors: A Tool for Detection of Plant Pathogens 67-90

(Aradhna Sagwal, Promil Kapoor, Satish Kumar and Abhishek Kumar)

4. RNA Interference: A Recent Approach for Plant Diseases

Management 91-104
(Sunil Kumar, Shivam Maurya, Stayadev Prajapati and Naresh Kumar)

5. Molecular Diagnostics Tools for Detection of Plant Pathogens 105-118

(Anil Kumar, Mohit Kumar, Mampi Suklabaidya and Rahul Patidar)

6. Role of Silicon in Plant Disease Management 119-129

(Jasmin Thomas)
 Chapter - 1
Host Defence Suppression by Plant Pathogens

Author
Meghana Suresh Nayak
M.Sc. (Agri.) in Plant Pathology

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Page | 2
 Chapter - 1
Host Defence Suppression by Plant Pathogens
Meghana Suresh Nayak

Abstract
Pathogens attack plants because during their evolutionary development
they have acquired the ability to live off the substances manufactured by the
host plants and some of the pathogens depend on the substances for survival.
Therefore, for a pathogen to infect a plant it must be able to make its way into
and through the plant, obtain nutrients from the plant and neutralize the
defence reaction of the plant. Plant defences pose selection pressure on
pathogens as a result they have evolved counter adaptations to suppress plant
defences.
Keywords: plant defence, pathogen, suppression
Introduction
Plants are nutritious and hence phytopathogens have specialized to attack
and consume them. In turn, plants have evolved adaptations to detect and
withstand these attacks. Such adaptations we call ‘defences’ and they can
operate either directly between the plant and the plant consumer or indirectly
i.e. when taking effect via other organisms. Plant defences put selection
pressure on plant-consumers and, as a result, pathogens have evolved counter-
adaptations to avoid, resist or manipulate plant defences.
(Alba et al., 2011)

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Five possible relationships between plants and potential pathogens

1) No relationship established between plant and the pathogen. For e.g.

fungal spore germinate but host doesn’t provide essential
requirements to penetrate.
2) A plant is antagonistic to the pathogen when it secretes inhibitory
compounds into its environment that prevents pathogen
development. For e.g. the stubble of some brassicas releases
'biofumigants' into the soil that prevent the hatching of nematode
eggs and inhibit the growth of some root-infecting fungi.
3) The pathogen is antagonistic to the plant when it secretes compounds
that damage the plant. Cochliobolus victoriae produces the toxin
victorin that causes severe seedling blight on susceptible cultivars of
oats, but has little effect on resistant cultivars or on other plant
species.
4) Mutual antagonism between plant and pathogen results in the
inhibition or death of both the host tissue and pathogen. For ex, an
incompatible interaction between the stem rust pathogen, Puccinia
graminis f. sp. tritici and resistant cultivars of wheat causes the death
of both host and pathogen cells.
5) Mutual adjustment leads to a compatible cellular relationship
between the host and pathogen. Symbiotic relationships between
mycorrhizal fungi and plant roots.
(David et al., 1996)

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Types of reaction developed by the plant against pathogen attack

1. Non-host resistance: Particular pathogen attacks only particular

host i.e. host specific. If there is no availability of the host, it can’t
penetrate so plant become resistant.
2. Horizontal/Polygenic resistance: Some plants produce both
resistant and susceptible genes. Susceptible genes cause infection but
plants don’t die.
3. Vertical/Monogenic/R resistance: Plant produce only one gene
which may be either resistant or susceptible.
Defence mechanism in plants
What is defence?
Plants have the capacity to defend themselves from the attack by various
pathogens. Different plants have different defence mechanism. Defensive
mechanism is genetic in nature.
• Structural or morphological barriers: Prevent entry of pathogen
into the host, acts as a physical barrier.
• Biochemical or induced barriers: Chemical or toxic substances
produced only after the pathogen attack.
Types of host defences
1. Constitutive defences: pre-infection.
2. Induced defences: post-infection.

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Constitutive defences
▪ Structural defence like wax and hairs act as water repellent, they
prevent the formation of water surface on the leaves where pathogen
can deposite and germinate. Thick cuticle provides more resistance
against pathogen attack. Shape and activity of stomata also acts as
defence like many pathogen enters the host through closed stomata
and if stomata are narrow and broad it provides resistance.
▪ Biochemical defence like some pathogens produce inhibitory
substances and phenolics which are toxic to the pathogen. For ex,
onion smudge produces phenolic substances like protocatechuic acid
and catechol. Red scaled onions are resistant to these phenolic
substances but white scaled onions are susceptible because they lack
these phenolic substances.
Induced defences
• Structural defence like formation of cork layer below the point of
infection which prevents the flow of toxic substances from infected
to healthy area. Abscission layer i.e. gap formed between infected
and healthy area. Gap is formed due to dissolution of middle lamella
of infected cells. Tyloses are the outgrowths formed in the protoplast
of xylem vessels which prevents flow of water. Deposition of gums
around the tissues acts as resistance mechanism.
• Biochemical defence like hypersensitivity-extreme case of
resistance. Phytoalexins are antimicrobial substances produced by
the host only after infection by pathogen like pisatin from peas,
phaseolin from beans etc acts as defence. Systemic acquired
resistance which is mediated through salicylic acid. It is systemic in
nature.

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Effectors
Protein factors produced by the pathogens which are mainly responsible
for causing infection and are also involved in suppression of plant defences.
Mechanism of effectors
1) Delivery of effectors into host cells.
2) Action of effectors in the apoplast.
3) Suppression of plant immunity.
4) Some effectors affect plant behaviour and development.
5) Molecular mimicry by effectors.
1) Delivery of effectors into host cells
Pathogens deliver effector proteins inside host cells through a different
mechanism like bacteria use Type Three Secretion System (T3SS) and fungi
use Haustoria to insert effector proteins into the host cell whereas nematodes
inject effector proteins inside vascular cell through stylet.
2) Action of effectors in the apoplast
Act as the plant-microbe interface, where they interfere with apoplastic
defenses (Van der Hoorn, 2008). For ex, effectors produced by Cladosporium
fulvum are Avr2, Avr9, Avr4 and ECP2 and Oomycetes-Phytophthora
infestans-secrete apoplastic effectors inhibit and protect pathogen against
plant hydrolytic enzyme.
3) Suppression of plant immunity
Contributes to virulence by suppressing basal defences and suppress
hypersensitive cell death elicited by various AVR proteins. For ex, Examples
Oomycete-RXLR effectors suppress host immunity and Phytophthora
infestans-Avr3a suppresses the hypersensitive cell death.
(Hogenhout et al., 2008)
4) Effectors affect plant behaviour and development

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 ▪ Pesudomonas syringae produces phytotoxin coronatine which
induces stomato to reopen so that pathogen can enter inside the host
cell and cause infection.
▪ Gibberella fujikuroi causes foolish seedling disease in rice which
produces gibberellin responsible for elongation of rice seedlings.
5) Molecular mimicry by effectors
The Pseudomonas syringae phytotoxin coronatine mimics jasmonoyl-
isoleucine which is a crucial plant signalling molecule for regulating plant
defence responses.
The role of selected effectors in plant defence suppression

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Apoplastic effectors of filamentous pathogens and their function

(Gunther et al., 2013)

Evolutionary process-central dogma of plant pathology
A) PAMP recognition triggers immunity-Resistance.
B) Effectors suppress immunity-Susceptibility.
C) R proteins recognise effectors activity-Resistance.
D) Effectors recognised by R protein is lost or modified-Susceptibility.

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 a) Recognition of pathogen-associated molecular patterns (such as
bacterial flagellin) by extracellular receptor-like kinases (RLKs)
promptly triggers basal immunity, which includes signaling through
MAP kinase cascades and transcriptional reprogramming mediated
by plant WRKY transcription factors.
b) Pathogenic bacteria use the type III secretion system to deliver
multiple effector proteins that target host proteins and suppress basal
immune responses, allowing significant accumulation of bacteria in
the plant apoplast.
c) Plant resistance proteins (R gene products, such as a TIR-NB-LRR
protein) recognize effectors activity and restore resistance through
strong Effector-triggered immune responses.
d) Pathogen avoids R gene-mediated defences by modifying or
eliminating the effectors that triggers those defences.
(Bent and Mackey, 2007)
Suppression of plant defences
a) Suppression of RNAi
A central defence against viruses is RNA interference (RNAi). RNAi is a
mechanism during which a plant generates virus-specific small RNAs that
form duplexes with viral nucleic acids which are subsequently degraded by
plant nucleases. Some of the viruses have the capacity to suppress RNAi
through the production of proteins that modify small RNA’s before they can
bind to their RNA target.

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 E.g. Cucumber mosaic virus

Cucumber Mosaic Virus produces suppressors that is CMV encoded 2b protein

which block the activity of the RNAi-specific nucleases.
(Zhang et al., 2006)
b) Suppression of local tissue death
Some pathogenic fungi produce so called supprescins of induced plant
defences. Supprescins are small glyco proteins that delay the transcription of
plant-genes involved in the production of toxins such as phytoalexins.

Tomato wilt caused by Fusarium oxysporum f. sp. lycopersici and

Septoria leaf spot caused by Septoria lycopersici secrete the enzyme
tomatinase that converts the defensive alkaloid tomatine of tomato Solanum
lycopersicum into harmless substances. Tomatine-hydrolysis products were
found to inhibit tomato defence signaling during infection showing the
detoxification and suppression.
(Pareja-Jaime et al., 2008)
c) Suppression via the jasmonate-salicylate antagonism
Pseudomonas syringae produces the JA-isoleucine-mimic-coronatine
that binds to the COI complex. COI-complex is activated by pathogen induced
JA-isoleucine initiating the degradation of transcriptional repressors of JA
dependent defence-genes there by allowing these genes to be transcribed there
by inhibiting the SA-dependent defence.

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 Mechanism of enzymes in pathogenesis: Enzymes are also involved in
the degradation of host defences.

Mechanism of toxins in pathogenesis: Toxins are antimicrobial

substances produced by the host cell only after the infection by pathogen and
are toxic to the pathogen.
1) The toxin will change the cell wall permeability: Cell wall is made
up of lipid and protein. Toxins are directly harmful for the synthesis
for lipid and proteins. Ex. Victorin, Lycomarasmin.
2) Disruption of normal metabolic processes: Increase in respiration
rate, malfunctioning of the enzymes. Ex. Pyricularin inhibits
polyphenol oxidase.
3) Inhibition of root growth: Fusarium moniliformae produce fusaric
acid which inhibit the root growth.
4) Affect the stomatal function
5) Less accumulation of phytoalexins: Oat produces phytoalexin i.e.
Avenalumin and toxin produced is Victorin. This toxin reduces the
action of avenalumin because it is mainly involved in defence
mechanism.
(Tsuge et al., 2013)

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Host specific toxins
Sl. No. Toxins Pathogens Diseases
01 Victorin Helminthosporium victoriae Victoria blight of oat
02 T-toxin Helminthosporium maydis Southern corn blight of maize
03 HC-toxin Helminthosporium carbonum Leaf spot of maize
04 HS-toxin Helminthosporium sacchari Eye spot of sugarcane
05 AK-toxin Alternaria alternata Leaf spot of pear
06 PM-toxin Phyllosticta maydis Corn spot
07 PC-toxin Periconia circinata Milo disease of sorghum

Non-host specific toxins

Sl. No. Toxins Pathogens Diseases
01 Fusaric acid Fusarium spp. Wilt
02 Tab toxin Pseudomonas syringae pv tabaci Wild fire of tobacco
03 Ophiobolin Helminthosporium oryzae Brown spot of rice
04 Cercosporin Cercospora spp. Leaf spots
05 Oxalic acid Sclerotium spp. Sclerotium rot
06 Pyricularin Pyricularia spp. Blast of rice
07 Ten toxin Alternaria spp. Leaf spot of sunflower

Defence effectors entering the host cell through several pathways

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 1) Prevent effectors from cell wall.
2) Pathogens prevent recognition receptors (PRR).
3) Plants secrete proteases (Pr) to degrade intercellular or extracellular
effector (IE or EE).
4) Pathogens secrete protease inhibitors to block the proteases activity.
5) Recognition of Pathogen Associated Molecular Patterns (PAMP’s)
by recognition receptors, which triggers PAMP triggered immune
response (PTI).
6) Recognition of EE through Trans-Membrane Leucine Rich
Receptors (TM-LRR)
7) Recognition of IE through Nucleotide Binding - Leucine Rich
Receptors (NB-LRR) leads to effector triggered immune response
(ETI).
8) Signalling events for both PTI and ETI are inhibited by IE, leads to
programmed cell death.
9) PTI and ETI involve transcriptional changes.
10) PTI and ETI also involve other responses like production of Reactive
Oxygen and Nitrogen Species (RONS).
Gebrie S.A. (2016)
Suppression of defence mechanism by biotrophic fungi
a) Cladosporium fulvum: Is a biotrophic fungal pathogen that causes
leaf mould of tomato. To avoid host recognition by host PRRs C.
fulvum secretes the effector Ecp6 that contains a LysM chitin binding
domain and also secretes the effector Avr2.
E.g. The Cladosporium fulvum virulence protein Avr2 inhibits host
proteases required for basal defence.
• Cladosporium fulvum produces ten effector proteins among which
four of these effectors are race-specific avirulence proteins (Avr2,
Avr4, Avr4E and Avr9), six are extracellular proteins (Ecp1, Ecp2,
Ecp4, Ecp5, Ecp6 and Ecp7).
• During growth in the apoplast, the fungus establishes disease by
secreting effector proteins Avr2 effector interacts with the apoplastic
tomato Cys protease Rcr3, which is required for Cf-2–mediated
immunity.

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Avr2-expressing Arabidopsis is more susceptible to the fungal pathogens
Botrytis cinerea and Plectosphaerella cucumerina

Typical necrotic symptoms caused by B. cinerea and P. cucumerina on

4-week old plants of three independent Avr2-expressing Arabidopsis lines
(At-Avr2-A to -C) at 4 DAI. On the progenitor Col-0 line and an Avr9-
expressing transgenic line (At-Avr9) are shown as controls developed no
symptoms.
Avr2-expressing tomato is more susceptible to Cladosporium fulvum race
2.

Adaxial side of MM-Avr2-A shows enhanced C. fulvum-induced

chlorosis compared with MM-Cf-0.
On the abaxial side, a pale yellowish shade around the main veins
indicative of fungal growth is observed on MM-Avr2-A and not on MM-Cf-
0.

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Avr2-expressing plants are more susceptible to Verticillium dahliae and
Botrytis cinerea

a) Typical stunted appearance of Avr2-expressing tomato leaves (MM-

Avr2-A) compared with the progenitor line (MM-Cf-0) upon
inoculation with B. cinerea at 3 DAI.
b) Microscopy observation of Avr2-expressing cleared tomato leaves
(MM-Avr2-A) compared with the progenitor line (MM-Cf-0) upon
inoculation with B. cinerea at 2 DAI after staining of fungal hyphae
and dead plant cells with Trypan blue.

a) Typical stunting induced by V. dahliae on three independent Avr2-

expressing Arabidopsis lines (At-Avr2-A to-C) compared with the
progenitor line (Col-0) at 2 weeks after inoculation.

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 Typical stunting induced by V. dahliae on three independent Avr2-
expressing Arabidopsis lines (At-Avr2-A to -C) compared with the progenitor
line (Col-0) at 2 weeks after inoculation.
(Esse et al., 2008)
Maize smut-Ustilago maydis
U. maydis effectors, Pep1, which is a small (178 amino acids) protein
required for successful invasion of epidermal cells of Zea mays. Inoculation
of leaves with deletion mutants of U. maydis lacking Pep1 led to the failure of
the pathogen to establish a compatible interaction with the host.
Pep1, a secreted effector protein of Ustilago maydis, is required for
successful invasion of plant cells
Identification of Pep1
The pep1 gene (um01987) resides on chromosome 3 of the U. maydis
genome. pep1 is not part of a gene cluster, i.e. upstream we find a putative
oxidoreductase (um01988) and downstream a sterol carrier (um01986), two
proteins not predicted to be secreted. The Pep1 protein comprises 178 aa and
is expected to be cleaved behind a putative N-terminal secretion signal.
Plant responses elicited by infection with SG200Δpep1

A) Macroscopic symptoms on maize leaves 4 dpi with SG200 and

SG200Δpep1. Red arrowheads mark necrotic regions in
SG200Δpep1 infected leaf tissue.

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 B) Papilla formation in maize cells attacked by SG200Δpep1.
Upper panel: Cell wall auto fluorescence. Lower panel: Bright field
projection of the same cell.
Expression and secretion of Pep1

A) Haploid sporidia of strain SG200pep1

B) SG200pep1 penetrating a maize epidermis cell; 24 hpi.
C) Intracellular growing hyphae of SG200pep1.
D) Tip of intracellularly growing hypha of SG200pep1 during cell to cell
passage. Pep1-GFP strongly accumulates at penetration sites.
E) Left panel shows SG200pep1 during cell to cell passage, 48 hpi.
Right panel shows the rupture of the cell wall of the same cell
inflicted by the penetrating fungal hyphae.
(Gunther et al., 2009)

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Powdery mildew in wheat and barley-Blumeria graminis
• B. graminis is an obligate biotrophic fungus.
• It grows as filamentous hyphae on the leaf surface.
• It forms a specialized feeding structures called a haustoria, which
surrounded by an intact plant plasma membrane.
• The AVRa10 and AVRk1effectors have important role infection
enhancement.
Flax (Linum usitatissimum) rust-Melampsora lini
• M. lini is a biotrophic fungus
• AvrL567, first identified flax rust effector protein was recognised by
the L6, L5 and L7 R protein.
• Other three secreted flax rust effector proteins, AvrM, AvrP123 and
AvrP4 have been identified, which have important role in host
defence suppression.
Case study 1
Tomatidine and lycotetraose, hydrolysis products of α-tomatine by
Fusarium oxysporum tomatinase, suppress induced defence responses in
tomato cells
Author: Ito et al. (2004).
Material and Method
Fungal pathogens of tomato produce extracellular enzymes, collectively
known as tomatinases, that detoxify the preformed antifungal steroidal
glycoalkaloid α-tomatine. Tomatinase from the vascular wilt pathogen of
tomato. Fusarium oxysporum f. sp. lycopersici cleaves α-tomatine into the
aglycon tomatidine (Td) and the tetrasaccharide lycotetraose (Lt).
The Lycopersicon esculentum L. cultivar ‘Ponderosa’ was used to
generate cell suspension cultures. Every 7 days, cells growing in the log phase
were transferred into fresh medium containing Murashige-Skoog salts and B5
vitamins supplemented with 0.5 mg/l 2.4-dichlorophenoxyacetic acid, 0.1
mg/l kinetin and 2% (w/v) sucrose, pH 5.7, and incubated under continuous
shaking at 100 rpm in the dark at 25 oC. Suspension cultured cells used for all
experiments were 3 days old. For obtaining tomato plantlets and cuttings,
seeds of tomato cultivar ‘Ponderosa’ were planted in minipots containing
vermiculite and maintained in a growth chamber at 25 oC with 16 h light and
8 h dark. F. oxysporum f. sp. raphani MAFF103058 (FOR#21) and F.

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oxysporum f. sp. lycopersici MAFF103036 (FOL#24) were grown on potato
dextrose agar at 25 oC.
Table 1: Histochemical staining of tomato cell suspension cultures with DAB to
visualize H2O2 accumulation

Result
Tomato cell suspension cultures were treated with fungal elicitor, a
mixture of fungal elicitor with 40 µM tomatidine, a mixture of fungal elicitor
with 40 µM lycotetraose, a mixture of fungal elicitor with 40 µM tomatidine
and 40 µM lycotetraose, and a mixture of fungal elicitor with 40 µM α-
tomatine, in the presence of DAB for 24 h. The reddish-brown coloration in
tomato cells treated with fungal elicitor (Elicitor) and with a mixture of fungal
elicitor with α-tomatine (Elicitor+Tomatine) at 12 h and 24 h incubation
indicates the accumulation of H2O2. In contrast, the reddish- brown coloration
was not observed in tomato cells treated with a mixture of fungal elicitor with
tomatidine (Elicitor+Tomatidine), a mixture of fungal elicitor with
lycotetraose (Elicitor+Lycotetraose) and a mixture of fungal elicitor with
tomatidine and lycotetraose (Elicitor+Tomatidine+Lycotetraose).

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 Table 2: Suppression of the elicitor-induced oxidative burst reaction in tomato cell
suspension cultures by tomatidine and lycotetraose

Result
Suppression of the elicitor-induced oxidative burst reaction in tomato cell
suspension cultures by tomatidine and lycotetraose. Suspension-cultured cells
of tomato were treated either with fungal elicitor alone or a mixture of fungal
elicitor with each 4 µM (s), 20 µM (j) and 40 µM of tomatidine (Td) and of
lycotetraose (Lt). The effects of Td and Lt on elicitor induced generation of
H2O2, the so-called oxidative burst. It has been known that the oxidative burst
commonly occurs in two distinct phases. As expected, two distinct phases of
the oxidative burst were observed: the first burst peaking at 20 min and the
more prolonged second burst peaking at 7 h after addition of elicitor. The
oxidative burst in tomato cultures was completely abolished by both
tomatidine and lycotetraose in a dose-dependent manner.
Table 3: Hypersensitive cell death of tomato cultured cells

Result
Suspension cultured cells of tomato were treated with fungal elicitor
alone, a mixture of fungal elicitor with 40 µM α-tomatine that with 40 µM
tomatidine or that with 40 µM lycotetraose. Tomato cells were stained with

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the viability stain Evans blue at a final concentration of 500 µg/ml for 10 min.
The cells were examined with a light microscope. Td and Lt inhibited the
elicitor-induced hypersensitive cell death (HCD) of tomato cultures.
Table 4: Effects of tomatidine and lycotetraose on infection of tomato cuttings with a
non-pathogenic strain of F. oxysporum f. Sp. raphani (FOR#21)

Result
Tomato cuttings were placed in a vial containing conidial suspension of
FOR#21in the presence of 40 µM tomatidine (#21+Td) or 40 µM lycotetraose
(#21+Lt) and incubated for 5 days. After the incubation, a 7-mm section of
hypocotyls was cut from right below the cotyledons, surface sterilized, placed
on a Fusarium-selective medium and then incubated for 4 weeks. Positive
control with a tomato pathogen F. oxysporum f. sp. lycopersici (#24) and
negative controls with no fungal inoculation (Control) or with no treatment of
tomatidine and lycotetraose (#21) are also performed.
FOR#21was observed around the ends of cuttings that had been exposed
to the fungus in the presence of either Td or Lt, indicating that the fungus had
colonized in the xylem vessels of tomato cuttings. No fungal colonies were
observed from the cuttings inoculated with FOR#21 in the absence of Td and
Lt during the 4-weeks incubation. Neither Td nor Lt promoted the growth of
FOR#21 on the medium in the absence of the plant materials. These results
suggest that Td and Lt would suppress defence responses of tomato cuttings
and thereby FOR#21, a non-pathogen of tomato was able to infect them.

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Plant defence suppression by bacterial pathogens

Plant defences targeted by pathogens to promote disease

The plant defences are targeted by plant pathogens. HR-based PCD is
activated by gene-for-gene strong recognition of pathogen Avr factors and is
a rapid response that functions to limit the establishment of pathogen
infection. Basal defences are elicited by the recognition of common pathogen
signatures, such as flagellin or chitin, or by weak recognition of Avr factors.
Basal defences result in the activation of defence gene expression or the
induction of late-onset cell death. Cell wall-based defences include cell wall
thickening and the formation of papillae near a nascent bacterial colony or
fungal penetration site. JA signalling activates defences that protect plants
from insects and necrotrophic pathogens. The activation of JA signaling
inhibits SA-dependent signaling and suppresses specific PR genes. Reactive
oxygen species (ROS) are directly toxic to bacteria and also act as signaling
molecules in plant immunity and PCD. Preformed antimicrobials, such as
saponins, are toxic to pathogens and function to repel pathogen attack. Genes
that encode acidic and basic PR proteins are activated by SA and JA-
dependent signalling pathways respectively and produce antimicrobial
compounds. PCD genes are required to signal and execute cell death and are
regulated in response to pathogen attack. The expression of the NHO1 gene is
activated by non-host and avirulent bacterial pathogens and is required in
some cases of non-host resistance.
(Robert et al., 2004)

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A model for the pathogen-mediated modulation of plant PCD
(Programmed cell death) during the course of disease

Pathogens such as DC3000, Phytophthora infestans may switch from

biotrophic to necrotrophic growth during the course of pathogenesis.
Experimental evidence suggests that the timing of host PCD is a crucial
determinant of disease outcome. Early HR-based PCD in the host leads to
resistance, whereas late-activated basal defences and PCD are observed during
disease. This model illustrates how a pathogen may modulate PCD to suppress
both HR-based and basal defences during pathogenesis.
a) Early in pathogenesis, the successful pathogen may need to suppress
HR-based PCD and basal defences to delay host detection and
establish infection.
b) Once infection is established, the pathogen may manipulate the host
to release the water and nutrients necessary for multiplication. With
time and increasing bacterial multiplication, however, the slowly
induced basal defences may become sufficiently activated to limit
pathogen growth effectively.
c) To overcome activated basal defences, to gain access to nutrients or
to aid dissemination, the pathogen may induce host PCD during late
infection. This cell death is seen as specks, spots or disease-
associated ‘necrosis’.

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Case study 2
Suppression of host defence in compatible plant–Pseudomonas syringae
interactions
Author: Kenya et al. (2005)
Infection cycle of Pseudomonas syringae

a) GFP-labeled Pst DC3000 (green rod-shaped) on the surface of an

Arabidopsis leaf.
b) An Arabidopsis stoma.
c) A scanning electron microscope image of the cross-section of an
Arabidopsis leaf infected by Pst DC3000. Rod-shaped bacteria
(indicated by arrows) multiply in the intercellular space outside of
host cells.
d) (Left) Arabidopsis and (right) bean plants infected by Pst DC3000
(Pto) or by P. syringae pv. phaseolicola (Pph). Arrows indicate
diseased leaf (chlorosis and necrosis) or pod (water-soaking).
e) (Left) Arabidopsis and (right) bean plants.
f) Migration of P. syringae to leaf surfaces.

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Suppression of basal plant defence

A diagram of the Arabidopsis thaliana-Pseudomonas syringae

interaction.
a) A scanning electron microscope image of the cross-section of an
Arabidopsis leaf infected by Pst DC3000.
b) Type III effectors and the toxin coronatine.
A model for the activation and inactivation of basal defences during Pst
DC3000 infection of susceptible Arabidopsis plants

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 PAMPs from TTSS-defective hrp mutants activate basal defence in an
SA-independent manner. In the case of flagellin, perception is mediated by the
FLS2 receptor, the signal is transduced via the MAPK3/6 pathway, and basal
defence is activated. In the CEL mutant, the AvrPto class of effectors
inactivates SA-independent basal defence. However, the host cell partially
overcomes the Avr Pto-mediated inactivation of SA-independent basal
defence and activates an SA-dependent basal defence pathway. The bacterial
factors involved in this SA-dependent activation are not known but they might
be type III effectors or the type III secretion process itself. In Pst DC3000,
HopPtoM and AvrE inactivate the SA-dependent basal defence and promote
disease-associated host cell death (i.e. necrosis).
RNA silencing mechanism-A defence mechanism against virus

RNA silencing is an adaptive defence mechanism that is triggered by

double stranded RNA (dsRNA). The process is initially triggered by dsRNA,

Page | 27
which can be introduced experimentally or arise from endogenous
transposons, replicating RNA viruses, or the transcription of transgenes. The
dsRNA trigger is cleaved by a ribonuclease III (RNAse III)-like enzyme
termed Dicer into 21–24 nucleotide duplexes termed short-interfering RNAs
(siRNAs). The production of siRNAs by Dicer is an ATP-dependent step and
probably involves interactions with other proteins, including an argonaute-like
protein, a dsRNA binding protein and an RNA helicase. The siRNAs produced
from a fully double-stranded RNA substrate by Dicer have distinctive
characteristics: they represent both polarities and have two nucleotide
3’overhangs with 5’phosphate and 3’hydroxyl groups. In another ATP-
dependent step, the siRNAs are denatured and incorporated into a multi-
subunit endonuclease silencing complex called RNA-induced silencing
complex (RISC). Within the activated RISC, single-stranded siRNAs act as
guides to bring the complex into contact with complementary mRNAs and
thereby cause their degradation.
(Braden et al., 2004)
Case study 3
Turnip crinkle virus coat protein mediates suppression of RNA silencing
in Nicotiana benthamiana
Author: Thomas et al. (2003)
Schematic representation of TCV genome organization

TCV is a member of the family Tombusviridae, genus Carmovirus. It has

a positive single-strand RNA encapsulated into an icosahedral capsid of 30
nm diameter. Its RNA encodes five open reading frames (ORFs) expressed
from the genomic and two sub genomic (sgRNA1 and 2) RNAs. Both P28 and
P88 are translated from the genomic RNA by read through of the P28 amber
termination codon and are involved in virus replication. The P8 and P9
proteins, synthesized from two overlapping ORFs of the 1.7 kb sgRNA1, are
required for virus cell-to-cell movement and systemic spread. The P38 capsid

Page | 28
protein is expressed from the 1.45 kb sgRNA2 and also plays a host-dependent
role in systemic movement.
Effect of potential suppressor activity on 35S-GFP expression after agro-
infiltration of Nicotiana benthamiana leaves

Leaves of N. benthamiana were agro-infiltrated with 35S-GFP (in

pBIN61), either in combination with pBIN61 (i.e., empty vector), or with
constructs expressing HC-Pro, TCV ORFs P8, P9, P28, P88, P38, ∆NP38 or
with 35S-TCV. Photographs were taken under UV light to show GFP green
fluorescence and the red fluorescence of chlorophyll. Photographs were taken
at 6 days post-infiltration when GFP expression had declined due to RNA
silencing except when a suppressor of silencing was present.
To identify the TCV suppressor, individual TCV ORFs were cloned into
a T-DNA expression cassette driven by the CaMV 35S promoter in a pBIN19
based vector (pBIN61) in A. tumefaciens strain C58C1. These clones were
infiltrated individually, but in combination with bacteria carrying 35S-GFP,
into leaf patches of non-transgenic N. benthamiana. For comparison, 35S-
GFP was also co-infiltrated with 35STCV, a cDNA clone of the complete
TCV RNA that generates a full infection in N. benthamiana or Arabidopsis
thaliana.
Result
In this assay, GFP was expressed transiently (over 2–5 days) in the
infiltrated patch and GFP was visualised as green fluorescence under UV light.
After six days only weak fluorescence is visible. This decline in expression
has been attributed to RNA silencing targeting the foreign gene expression.
Hence, in the presence of a suppressor such as the potyvirus HC-Pro, GFP
expression remained high and was visualized as bright green fluorescence.
Since the silencing of GFP was established de novo, the assay had the potential
to identify suppressors of initiation and or maintenance of RNA silencing.

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 For TCV, the P8, P9 and P38 (coat protein; CP) had all been implicated
as disease determinants. Neither of these proteins showed suppressor activity.
Similarly, no activity was seen following co-expression of the replicase-
related products P28 or P88 with GFP. In contrast, co-expression of the TCV
P38 with GFP produced intense green fluorescence which persisted for at least
22 days. Considering the need for TCV P38 to produce capsids in infected
cells it was surprising to see, however, that co-infiltration of 35S-GFP and
35S-TCV showed barely any change in GFP fluorescence, compared to
infiltration with 35S-GFP alone.
Phenotypic effects associated with the expression of TCV P38 from the
PVX vector

Plants were agro-inoculated with PVX vector constructs containing no

insert (wild-type; wt), the TCV P38 cDNA (P38), an untranslatable P38
sequence (mCP), or an N-terminally deleted version of P38 (∆NP38). After 6
days tissues inoculated with PVX-P38 showed severe necrosis while the other
infections were asymptomatic. After 10–14 days, plants infected with PVX-
wt, PVX-mCP or PVX-∆NP38 showed the typical chlorotic symptoms of
PVX infection. Plants infected with PVX- ∆ NP38 showed near-complete
necrosis.
Plant defence suppression by nematodes
• Plant parasitic nematodes are microscopic worms.
• These obligate endo parasites have a biotrophic relationship with
plants, in which they induce the differentiation of root cells into
hypertrophied, multinucleate feeding cells (FCs).
• Effectors synthesized in the esophageal glands of the nematode are
injected into the plant cells via the syringe-like stylet and play a key
role in manipulating the host machinery.
(Quentin et al., 2013)

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Nematode effectors target host functions to establish feeding cells

Case study 4
A novel nematode effector suppresses plant immunity by activating host
reactive oxygen species-scavenging system
Author: Lin et al. (2016)
Evidence is emerging that plant-parasitic nematodes can secrete effectors
to interfere with the host immune response. A novel effector, MjTTL5 could
suppress plant immune response.
Expression patterns of MjTTL5 in Meloidogyne javanica

Page | 31
 a) Immunolocalization of MjTTL5 in a pre-parasitic second-stage
juvenile nematode (pre-J2) incubated with anti-MjTTL5 serum,
showing the protein located in subcentral glands (left panel) and a
pre-J2 incubated with pre-immune serum showing no signal (right
panel).
b) Developmental expression pattern of MjTTL5-Transcriptional
expression of MjTTL5 was analyzed by RTqPCR at different
developmental stages of M. javanica. The results showed that the
expression of MjTTL5 was increased and reached a peak in the early
par-J2 at 2 dpi. Subsequently, the expression of MjTTL5 was
gradually reduced and reached a basal level at the female stage.
MjTTL5 plays a role in the early stages of nematode parasitism.
Expression of MjTTL5 in Arabidopsis enhances the susceptibility to
Meloidogyne javanica

a) Transgenic Arabidopsis expressing MjTTL5 showed enhanced

susceptibility to M. Javanica-The susceptibility of these transgenic
Arabidopsis lines to nematode infection was then determined, and the
results showed that all three transgenic lines were significantly (P <
0.05) more susceptible to M. javanica infection than wild type
Arabidopsis, as evidenced by the statistically significant higher
number of females inside the roots at 42 dpi.
b) Photograph of representative transgenic and wild-type (WT) plants-
The transgenic lines resulted in stunted growth as compared to the
wild type.

Page | 32
MjTTL5 suppresses flg22-mediated reactive oxygen species (ROS)
production in Nicotiana benthamiana

Result
Agrobacterium tumefaciens strain GV3101 derivatives carrying MjTTL5,
MjCBP (cellulose-binding protein) or hemagglutinin (HA) constructs were
infiltrated into the leaves of 3-wk-old N. benthamiana plants. Infiltrated leaf
discs were collected 48 h post agroinfiltration and assayed for ROS
production. The results showed that in planta expression of MjTTL5
drastically reduced the flg22-induced ROS production in comparison with the
controls. The attenuated defence gene induction and decreased ROS burst in
response to flg22 treatment indicate that MjTTL5 plays a role in suppression
of host plant PTI.

Page | 33
MjTTL5 alters the rate of H2O2 accumulation

Three independent Arabidopsis lines expressing MjTTL5 showed a

significantly lower amount of H2O2 than wild-type (WT) plants.
References
1. Alba JM, Glas JJ, Schimmel BCJ, Kant MR. Avoidance and suppression
of plant defences by herbivores and pathogens. J Pl. Interact. 2011;
6(4):221-227.
2. Bent AF, Mackey D. Elicitors, effectors and R genes: The new paradigm
and a lifetime supply of questions. Annu. Rev. Phytopathol. 2007;
45:399-436.
3. Braden MR, Gail JP, Vicki BV. Plant viral suppressors of RNA silencing.
Virus Res. 2004; 102:97-108.
4. Esse HPV, John W, Melvin DB, Yadeta KA, Baarlen P, Boeren S et al.,
The Cladosporium fulvum virulence protein avr2 inhibits host proteases
required for basal defence. Plant Cell. 2008; 20:1948-1963.
5. Gebrie SA. Biotrophic fungi infection and plant defence mechanism. J
Plant Pathol. Microbiol. 2016; 7(9):1-6.
6. Gunther D, Hemetsberger C. Apoplastic immunity and its suppression by
filamentous plant pathogens. New Phytol., 2013, 16.

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7. Gunther D, Linde KV, Daniela A, Daniela S, Alexander H, Mohanty A et
al., Pep1, a Secreted Effector Protein of Ustilago maydis, Is Required for
Successful Invasion of Plant Cells, 2009.
8. Hogenhout SA, Van der Hoorn RAL, Terauchi R, Kamoun S. Emerging
Concepts in Effector Biology of Plant-Associated Organisms. Mol. Plant
Microbe Interact. 2008; 22(2):115-122.
9. Ito S, Eto T, Tanakaa S, Yamauchia N, Takaharab H, Ikeda T. Tomatidine
and Ly cotetraose, hydrolysis products of α-tomatine by Fusarium
oxysporum tomatinase, suppress induced defense responses in tomato
cells. FEBS Lett. 2004; 571:31-34.
10. Lin B, Zhuo1 K, Chen S, Hu1 L, Sun L, Wang X et al., A novel nematode
effector suppresses plant immunity by activating host reactive oxygen
species-scavenging system. New Phytol. 2016; 209:1159-1173.
11. Nomura K, Melotto M, Yang SH. Suppression of host defence in
compatible plant-Pseudomonas syringae interactions. Curr. Opin. Pl. Bio.
2005; 8:361-368.
12. Pareja-Jaime Y, Roncero MIG, Ruiz-Roldan MC. Tomatinase from
Fusarium oxysporum f. sp lycopersici is required for full virulence on
tomato plants. Mol. Plant-Microbe Int. 2008; 21:728-736.
13. Quentin M, Abad P, Favery B. Plant parasitic nematode effectors target
host defence and nuclear functions to establish feeding cells. Front. Plant
Sci. 2013; 4:1-7.
14. Thomas CL, Leh V, Lederer C, Maule AJ. Turnip crinkle virus coat
protein mediates suppression of RNA silencing in Nicotiana
benthamiana. Virol. 2003; 306:33-41.
15. Tsuge T, Harimoto Y, Akimitsu K, Ohtani K, Kodama M, Akagi Y et al.
Host-selective toxins produced by the plant pathogenic fungus Alternaria
alternata. FEMS Microbiol. Rev. 2013; 37(1):44-66.
16. Zhang XR, Yuan YR, Pei Y, Lin SS, Tuschl T, Patel DJ et al., Cucumber
mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1
cleavage activity to counter plant defence. Gen. Dev. 2006; 20:3255-
3268.

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Page | 36
 Chapter - 2
Management of Plant Viruses

Authors
Sompalli Suresh Rao
Ph.D. Scholar, Department of Plant Pathology, S.V.
Agricultural College, Tirupati, Andhra Pradesh, India
R. Sarada Jayalakshmi Devi
Professor and University Head, Department of Plant Pathology,
S. V. Agricultural College, Tirupati, Andhra Pradesh, India
K.V. Hari Prasad
Associate Professor, Department of Entomology, S.V.
Agricultural College, Tirupati, Andhra Pradesh, India
M.K. Jyosthna
Associate Professor, Department of Plant Pathology, S.V.
Agricultural College, Tirupati, Andhra Pradesh, India
P. Arunasri
Assistant Professor, Department of Plant Pathology, S.V.
Agricultural College, Tirupati, Andhra Pradesh, India

Page | 37
Page | 38
 Chapter - 2
Management of Plant Viruses
Sompalli Suresh Rao, R. Sarada Jayalakshmi Devi, K.V. Hari Prasad, M.K. Jyosthna and
P. Arunasri

Abstract
In the countries embedding tropical and subtropical conditions the
incidence of plant virus diseases and their damage losses caused to
economically important crops are reportedly increasing every day. The
frequent emergence of new viral diseases is mainly due to international
trade, climate change, and the ability of viruses for rapid evolution.
Integrated management approaches involving utilization of virus resistant
crops and efficient management of insect vectors can reduce this disastrous
problem. In developing countries such strategies are rarely applied due to
lack of farmer’s knowledge about plant virus diseases. Therefore, starting
with the farmer known cultural practices here, discussed about all the
possible prophylaxis to restrain virus dispersion management techniques like
Quarantine approaches, Biological control, Insect vector management by
highlighting the Viral resistant mediated techniques like coat protein,
moment protein and replicase protein mediated resistance and also the
RNAi, CRISPR management strategies to apply against plant viruses
effectively. Disease management relies strongly on a fast and accurate
identification of the causal agent.
Keywords: virus diseases, ecology, management, cultural, biological, vector
control, coat protein, moment protein, replicase protein resistance, RNAi,
CRISPR
Management of plant viruses
I. Ecology
The factors influencing the behaviour of a virus in a given physical
situation include host range, tissue tropism, pathogenesis, and host
responses. It is a fundamental concept based on the relational properties of
the virus and is not a property of the environment. Effect of climatic
conditions on viruses: It includes light, temperature, nutrition. Certain

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viruses prefer high temperatures while some are virulent at lower
temperature.
• Cocomo virus-high temperature (more rapid symptoms).
• N. glutinosa when inoculated with TMV at 20 degrees it shows
necrotic local lesions while at 30 degree it show systemic mosaic.
Effect of cropping systems
• Maize when introduced in to Africa suffered severely form masteri
virus transmitted by leaf hopper.
• South American cacao when introduced into west Africa suffered
from cacao swollen shoot badna virus.
• Modern crop improvement practices include increase in cropping
intensity, change in time of planting, introduction of year-round
cultivation etc…,
• Rice viruses were reported in 1950 from traditional variety of rice
(not much severe) but in 1960-became severe because of HYV
introduction. (Rice tungro).
Change of crop genetic makeup
• Introduction of new cultivars-risks outbreak of new virus diseases.
• Yellow mottle caused by rice yellow mottle (sobemo) virus
endemic in Africa, has become a predominant rice disease there
after the introduction of Asian rice genotypes.
• Suddenly a new virus disease may arise because of overlooked
introduction of susceptibility to viruses.
Crop plants
• Infection often originates within the crop itself, particularly if
grown from propagation material that is partially infested with
infection propagules.
• Cucumber green mottle mosaic virus - Seed borne etc.
• In annual crops infection may come from surviving ground keepers
or volunteers.
• Grapevine roots may survive in vineyard soil for several years,
during which nematodes may transmit nepo virus from them.
Weeds
• Acts as reservoir of viruses. A number of viruses may hibernate and
spread in seeds of wild plants.

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 • Seeds of chick weed acts as reservoir of Cucumo virus these seeds
remains viable in soil for 10 years and virus is retained as long as
they remain viable.
Other sources
• Certain plant viruses may be retained in overwintering vectors if
persistent or in a few instances in the eggs of insects or in the
resting spores of fungi.
• Tobacco stunt virus was found to survive in resting spores of
Olpidium brassicae for more than 20 years.
• Stable viruses may remain infectious in soil and water and be
mechanically introduced into plants through wounds.
II. Management of virus
There are different methods for the management of virus
1) Cultural methods
2) Quarantine methods
3) Chemical control methods
4) Resistance
5) Biological methods of control
6) Integrated Methods of disease management
1. Cultural control methods
These methods includes:
a) Field sanitation
b) Crop rotation
c) Green manuring
d) Tillage practices
e) Trap crop
f) Special cultivation
g) Soil sterilization
a) Field sanitisation
• Field sanitation means making field surroundings clean by
removing weeds, alternate hosts etc.,
• Some virus diseases are carried in weeds and some are weed borne
diseases.
• E.g.: Prostate pig weed-potato virus y.

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b) Green manuring
• It is the practice of incorporating green biomass of plant species
into a field either by growing the crop in the same field or by
bringing the crop residues from outside.
• It decreases soil infestation with pathogenic fungi, bacteria, virus by
increasing microbial activity.
• Crops chosen should have short growth cycle.
c) Tillage
• It enhances the degradation of plant materials after harvest by
mixing with soil and inverts the sub soil to the surface.
• It helps in reducing weeds, insects, pathogens.
d) Trap crop
• Generally trap crops are raised to control insects and nematodes that
act as vectors for virus transmission.
• Plant with root exudates that stimulate egg hatch but in which
nematode reproduction does not occur.
• A trap crop can reduce nematode population.
Special cultivation
• Flooding in paddy helps in weed control.
• Some weeds act as vectors for rice tungro. Flooding reduces rice
tungro.
Soil sterilization
Physical methods include soil solarization and hot water treatments. Soil
can be sterilized in green houses and sometimes in seed beds by aerated
steam or hot water. At about 500C, nematodes, some oomycetous fungi and
other water molds are killed. At about 60 and 720C, most of the plant
pathogenic fungi and bacteria are killed. At about 820C, most weeds, plant
pathogenic bacteria and insects are killed. Heat tolerant weed seeds and
some plant viruses, such as TMV are killed at or near the boiling point (95-
1000C).
Hot water or hot air treatment
Hot water treatment or hot air treatment will prevent the seed borne and
sett borne infectious diseases. Hot water treatment of certain seeds, bulbs
and nursery stock is done to kill many pathogens present in or on the seed

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and other propagating materials. Hot water treatment is used for controlling
sett borne diseases of sugarcane [whip smut, grassy shoot and red rot of
sugarcane (52 oC for 30 min)].
2. Preventing the introduction of virus
a) Quarantine and seed certification.
b) Restricted planting through legislation.
c) Heat therapy for virus free material
d) Tuber indexing in potato
e) Tissue culture techniques.
a) Quarantine
• Very important for the management of virus diseases.
• Plant quarantine laws were first enacted in France (1660), followed
by Denmark (1903) and USA (1912).
• In India, 16 plant quarantine stations are in operation by the
“Directorate of plant protection and quarantine” under the ministry
of food and agriculture, government of India.
• Plant quarantine measures are of 2 types.
1) Domestic quarantine-Bunchy top of banana, Banana mosaic etc.,
2) International quarantine.
b) Restricted planting through legislation
• Legislation may restrict the growing of those crops which are likely
to become infected with virus diseases or act as sources of survival
of diseases for other crops. E.g.: Bunchy top of banana.
c) Heat therapy
• Viral disease transmission is through cuttings, bulbs, grafts.
• Sugarcane mosaic has been controlled by treating the cuttings at 53
- 54 degrees.
• Low temperatures of 5 to 15 degrees have been used to eradicate
potato virus y.
d) Tuber indexing in potato
• Used for production of virus free tubers.
• It is done to discard virus infected clones.
• ELISA has been employed for tuber indexing.

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3. Control of vectors
This is one of the important steps in the prevention of plant virus
diseases during growth of the crop.
Insecticides
• Substances that kill insects by their chemical actions are called
insecticides.
I. Based on the mode of entry of the insecticides into the body of the
insect, they are groups as
1. Contact poisons
2. Stomach poisons
3. Fumigants
4. Attractants
5. Repellents
1. Contact poisons
These insecticides are capable of gaining entry into the insect body
either through spiracles and trachea or through the cuticle itself. Hence, these
poisons can kill the insects by mere coming in contact with the body of the
insects. E.g. DDT and HCH.

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2. Stomach poisons
The insecticides applied on the leaves and other parts of plants when
ingested act on the digestive system of the insect and bring about the kill of
the insect. E.g.: Calcium arsenate, lead arsenate.
3. Fumigants
A fumigant is a chemical substance which is volatile at ordinary
temperatures and sufficiently toxic to the insects. Fumigation is the process
of subjecting the infested material to the toxic fumes or vapours of chemicals
or gases which have insecticidal properties. Chemical used in the fumigant
and a reasonably air tight container or room is known as fumigation chamber
or “Fumigatorium”. Fumigants mostly gain entry into the body of the insect
through spiracles in the trachea.
Commonly used fumigants and their doses
1) Aluminium phosphide, marketed as Celphos tablets used against
field rats, groundnut bruchids etc.
2) Carbon disulphide 8-20 lbs/1000cft of food grains.
3) EDCT (Ethylene Dichloride Carbon Tetrachloride) 20-30
lbs/1000cft of food grains.
4) EDB Ethylene dibromide 1 lb/1000cft of food grains.
5) SO2: By burning sulphur in godowns SO2 fumes are released.
4. Attractants
Chemicals that cause insects to make oriented movements towards their
source are called insect attractants. They influence both gustatory (taste) and
olfactory (smell) receptors (or) sensilla.
Types of attractants
1) Pheromones
2) Natural food lures
3) Oviposition lures
4) Poison baits
1) Pheromones
In 1959 Karlson and Butenandt coined the term pheromone. For a
chemical that is secreted into the external environment by an animal and that
elicits a specific response in a receiving individual of the same species. It is
also referred to as “ectohormone”. Depending on their mode of action
pheromones are divided into two general classes.

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 i) One which gives a releaser effect: An immediate and reversible
behavioural change is produced in the receiving animal.
ii) One which gives a primer effect: A chain of physiological
changes is triggered off in the receiving animal. E.g.: Gustatory
stimulation, controlling caste determination and reproductive
control in social Hymenoptera (Ants and Bees), Isoptera (Termites).
Behaviour: Releasing pheromones are typically odorous and act
directly on the central nervous system of the receiving animal. E.g.: Alarm,
trail following, aggregation for mating, feeding (or) oviposition, the
pheromones that promote aggregation are sex pheromones and aggregation
pheromones.
a) Sex pheromones
A Sex pheromone released by one sex only triggers off a series of
behavior patterns in the other sex of the same species and thus facilitates
mating. The male insects respond to the odorous chemical released by the
female. In certain species of insects the males are known to produce the sex
pheromone which attracts the females.
Ex: In the cotton boll weevil Anthonomus grandis.
The sex pheromones are specific in their biological activity, the males
responding only to a specific pheromone of the female of the same species,
and their reactions are directed towards the air currents carrying the odour.
The time of release of the pheromones by the females and response by male
to them appears to be specific for each species. Effective distances for sex
pheromones depend on the threshold concentration for male stimulation and
release rate from the female.
The following sex pheromones have been isolated and identified.
Bombycol: Silkworm, Bombyx mori.
Gyplure: Gypsy moth, Perthetria dispar.
Gossyplure: Pink bollworm, Pectinophora gossypiella.
Trimedlure: Mediterranean fruits, Ceratitis capitata.
Cuelure: Melon fly, Bactrocera cucurbitae.
Litlure: Tobacco cutworm, Spodoptera litura.
Helilure: Red gram pod borer, Helicoverpa armigera.
Amlure: Chaffer beetle, Amphimallon sp.

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 Looplure: Cabbage looper, Trichoplusia ni.
Ferrolure: Coconut Red Palm Weevil, Rhynchophorus ferrugineus.
Lucilure: Brinjal Shoot and Fruit Borer Leucinodes orbonalis.
Sex pheromones in insect pest management
1) Monitoring of insect pests: Traps baited with synthetic sex
pheromones is useful in estimating population and detecting early
stages of pests. Four pheromone traps per acre is recommended.
2) Mass-trapping: (Male annihilation technique): Large number of
pheromones baited traps can be used in the fields to capture male
moths of newly emerged and reduce the number of males for
mating.
3) Control of pest by mating disruption: By permeating the
atmosphere with higher concentration of the pheromone the
opposite sex is rendered confused and unable to locate their mates.
b) Aggregation pheromones
The pheromone released by one sex only elicits response in both sexes
of a species. In scolytid (or) bark beetles the males secrete the pheromone
into the hind gut which gets incorporated in to the faceal pellets and through
them attracts flying males and females towards the galleries. In Trogoderma
granarium mixture of fatty acid esters and methyl and oleate function as
aggregation pheromones.
c) Trial marking pheromone
At low concentrations mostly used by foraging ants and white ants. In
ants Formica rupa, formic acid while termites, Zootermopsis nevadensis
hexanoic acid functions as the trial marking pheromone.
d) Alarm pheromones
These substances are elaborated by mandibular glands, sting apparatus,
anal glands which typically results in fight or aggression. Dolichoderine ants
release a fruity odour by the worker that results in a erratic behaviour of
workers, when this is discharged into mandibles onto an intruding insects
that becomes marked as aggressor.
2) Natural food lures
These are Chemicals present in plant and animal hosts that attract (lure)
insects for feeding. They stimulate olfactory receptors and may be

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 1) A floral scent in case of the nectar feeding insects
2) Essential oils for the phytophagous insects.
3) Decomposing products for the scavenger
4) Carbon dioxide, lactic acid and water for the blood sucking insects.
3) Oviposition lures
These are chemicals that govern the selection of suitable sites for
oviposition by the adult female for example, P-methyl acetophenone attracts
adult female rice stem borers to oviposit.
5. Poison baits
Poison baits are a mixture of food lures and insecticides. The effort is
made to make the bait more attractive to insects than their natural food and
also a smaller quantity should be able to attract the largest number of insects.
Baits are used when for some reason spraying (or) dusting of insecticides is
not practicable. For instance, when insects live hidden under the soil, inside
the fruits and vegetables (or) for household insects like ants, cockroaches
and houseflies.
6. Repellents
Repellents are broadly classified as Physical repellents and Chemical
repellents.
1) Physical repellents
These produce repellence by physical means and are of the following
kinds.
i) Contact stimuli repellents
These are substances (Such as dusts, granules, water, oils, leaf hairs,
spines and waxes) that influence the surface texture of the plants to produce
a disagreeable effect on the tactile sense of the insects.
ii) Auditory repellents
Theses employ sound to ward off insects. For instance, an amplified
sound has been found effective in repelling mosquitoes, pyralid moths and
files.
iii) Visual repellents
White light normally attracts insects but the yellow colour light is the
least attractive and to some extent acts as a visual repellent to insects.

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iv) Excitatory repellents
Chemicals such as pyrethrum, DDT, BHC etc., which excite the insects
tarsi by stimulating the sensory nerves and force them to leave the treated
surface.
v) Feeding repellents
Substances that inhibit feeding in insects are called feeding repellents
(or) Antifeedent.
2. Chemical repellents
These are chemicals that affect tactile, olfactory (or) gustatory receptors
of insects and could be plant origin (or) synthetic as follows.
i) Repellents of plant origin
The oil of citronella remained a commonly used mosquito repellent and
still continues to be a constituent of a popular brand of commercial mosquito
repellent, Odomos. The oil is extracted from the lemon grass, Cymbopogon
nardus and contains citronellol, geraniol (as the main constituents), borneol
and terpenes (in small amounts) of which the first two are regarded as the
main repellents for mosquitoes. Pyrethrum is another plant product which
not only acts as an insecticide but in low concentrations, also as a repellent
for blood-sucking insects. Clothes impregnated with some pyrethroids have
been found to afford protection against the attack of many insects vectors of
diseases like Aedes aegypti, Anopheles quadrimaculatus etc.,
ii) Synthetic repellents
Diethyl toluamide, protects the bearer against mosquitoes, ticks, fleas
and biting files. The others Bordeaux mixture, Dimethyl phthalate and
Indalone acts as repellents for insects.
Uses of repellents
Repellents can find several uses such as
1) They can be used on the body in some formulation to ward off
insects.
2) They can be used as fumigants in an enclosed area of insects.
3) They can be used as dusts and sprays on domestic animal to protect
them from noxious biting and blood-sucking insects.
4) They can be used to drive insects from their natural breeding
grounds to areas treated with an insecticide (or) a chemosterilant to
kill (or) sterilize them.

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Insect antifeedants
Antifeedant is a chemical that inhibits feeding but does not kill the
insect directly; the insect often may remain on the treated plant material and
possibly may die of starvation. These are also caused as “Feeding
deterrents”. There are three main sites for the sense of taste in insects located
in the mouth, on the tarsi and on the antennae.
4) Biological control
• Parasites and predators undoubtedly play a major role in limiting
the population growth of aphids and other insects.
• This method of control is of great importance because it is
economical, beneficial and simple.
• About 40-60% of aphids species are eaten by predators belonging to
coccinellid beetles.
• Nematophagous fungi: Endoparasitic fungi-Nematophthora.
• Predaceous fungi: Arthrobotrys conoides, Oligospora, Dactylella
doedycoides, Dactylaria candida
• Opportunistic fungi: Colonize nematodes.
E.g.: Paecilomyces lilacinus, Verticillium or Pochonia chlamydosporia,
Hirsutella sps.
• Mycorrhiza: Glorris fasiciculatum
• Bacteria: Pasteuria penetrans-Pseudomonas fluorescens.

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5) Virus resistance
• Resistance can refer to the capacity of a plant to less the activity or
harmful effects of a pathogen or pathogens such as virus, fungus,
bacterium.
• Comparatively very few viruses have been controlled in this manner
Satisfactory control have been achieved in sugarcane mosaic
disease.
• CPRI hybrids in potato namely VP-8, PH-12-22, 8-5, 55-123
Resistant to pv-y and pv-x.
• Resistance to vector aphids has been observed in potato cultivars
like Kufri badshah, kufri Shakti, kufri jyoti.
• Tolerant varieties kufri ashoka, kufri badshah, kufri sindhuri are
tolerant against pv-x and pv-y.
B. Host plant derived resistance
• Many plant genes have been reported and identified to impart
resistance against viruses.
• A naturally occurring gene from tobacco are introduced in tomato
has been reported to give good protection against TMV and Tomato
mosaic virus.
Virus replication in plant cell
Steps in virus replication
1) Virus attachment to plant cell
2) Release of viral genome into cytoplasm (if RNA) or nucleus (if
DNA).

Page | 51
 3) Translation of viral replicase, other factors like chaperones,
movement proteins, regulators and coat proteins coded by virus
genome.
4) Replication of genome by viral replicase or host replication
machinery.
5) Assembly of virus particles, release from plant cell.

Virus resistance
I. Expression of viral proteins in plants
1) Coat protein
2) Replicase
3) Movement protein

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II. Expression of RNA
1) Interfering RNA.
2) Non-coding RNA sequences.
III. Expression of plant defence genes or other non-viral genes
1) R genes.
2) Ribosome-inactivating proteins.
3) Protease inhibitors.
4) RNA modifying enzymes.
5) Single-chain variable fragment (scFv) antibodies.
1. Coat-protein mediated virus resistance
Coat protein-mediated resistance (CP-MR) refers to the resistance of
transgenic plants that produce CP to the virus from which the CP gene is
derived. May arise due to:
1) Interaction between transgene CP and virus CP.
2) Binding of transgenic CP to host factors required for disassembly of
virus.
3) Interaction of CP with nuclear inclusion protein b (specific to
Potyviruses).

• Computer-assisted graphic representation of a model that shows the

predicted interactions between transgenic TMV CP (orange, upper)
and challenge virus (grey, lower) that are involved in coat-protein-
mediated resistance (from Bendahmane & Beachy 1998).

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Cross protection
Transgenic plants expressing coat protein showed cross protection to
infection by other closely related viruses. This is known as Cross protection.

2. Replicase mediated virus resistance

• Engineering virus resistance by using genes encoding viral RNA
dependent RNA-polymerases (RdRps) was first reported for TMV.
• Interactions between transgenic replicase proteins and other virus-
encoded proteins may affect the processes of replication and cell-to-
cell movement.
• Interactions between truncated (non-functional) transgenic replicase
may arise due to RNA-mediated silencing.
• Interactions between RNA of transgenic replicase and virus-
encoded RNA may lead to RNA-mediated silencing of virus
replicase.
3. Movement protein mediated resistance
• Movement Proteins (MP) interact with plasmodesmata or form
tubules to allow intercellular trafficking of virions.

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 • Transgenic plants expressing mutant MP show resistance through
competition for plasmodesmatal binding sites.
• Resistance is against a broad spectrum of viruses, since most
viruses use MPs for intercellular movement.

4. RIP-Ribosomal inactivating protein

• Ribosome-inactivating proteins (RIPs) are toxic N-glycosidases that
depurinate eukaryotic and prokaryotic rRNAs, thereby arresting
protein synthesis during translation.
• RIPs are widely found in various plant species and within different
tissues.
• in vitro and in transgenic plants that RIPs have been connected to
defense by antifungal, antibacterial, antiviral and insecticidal
activities.
• RIPs are three different types based on their physical properties.
• Ribosomal inactivating proteins (RIP) catalyse depurination of
ribosomes and show antiviral activity.
• Pokeweed antiviral protein (PAP) is stored in the cell wall matrix of
leaf mesophyll cells and enters cytoplasm only in response to injury
and inactivates ribosomes.
• Transgenic tobacco plants expressing PAP showed antiviral activity
due to inactivation of ribosomal activity in the infected cells by
PAP entry into the cytoplasm.

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5. RNA Interference (RNAI)
• RNA interference (RNAi) is a highly evolutionally conserved
process of post-transcriptional gene silencing (PTGS) by which
double stranded RNA (dsRNA).
• When introduced into a cell, causes sequence-specific degradation
of homologous mRNA sequences.
• It was first discovered in 1998 by Andrew Fire and Craig Mello in
the nematode worm Caenorhabditis elegans and later found in a
wide variety of organisms, including mammals.
• RNA INTERFERENCE Phenomena first observed in petunia
• Attempted to overexpress chalone synthase (anthocyanin pigment
gene) in petunia. (trying to darken flower color) Caused the loss of

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 pigment Called co-suppression because suppressed expression of
both endogenous gene and transgene.

Steps
• The RNAi pathway can be divided into three major.
• First is the conversion of dsRNA input into 21-23bp small
fragments by the enzyme Dicer.
• Secondly the loading of small RNAs into large multiprotein
complex RISC.
• Lastly the sequence specific silencing of the cognate gene by RISC
that is guided by the small RNA fragment.

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Dicer
• Double-stranded RNA triggers processed into siRNAs by enzyme
RNAseIII family, specifically the Dicer family
• Dicer family proteins are ATP-dependent nucleases.
• These proteins dice long dsRNA into small RNA duplexes of 21-
26nt sizes.
• Loss of dicer: loss of silencing process
RISC
• RISC has helicase, exonuclease, endonuclease and homology
searching proteins.
• Initial RISC is inactive until transformed into active form by
unwinding of the siRNA duplex and loss of sense (passenger)
strand.

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 • Antisense (guide) strand defines specificity of RNAi.
• The transactivating response RNA-binding protein (TRBP) is a
protein with three double-stranded RNA-binding domains.

6. Micro RNA (miRNA)

• MicroRNA’s (miRNA’s) are small noncoding RNA molecules that
regulate eukaryotic gene expression at the translation level.
• miRNA is a ssRNA of ~22 nucleotides in length.
• Generated by the RNase-III-type enzymes Drosha and Dicer from
an endogenous transcript that contains a local hairpin structure.
• pri-miRNAs contain cap and poly(A) tail and are transcribed by
RNA Polymerase II.
• Created by similar process to siRNA.
• Generally prevents binding of ribosome.

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7. CRISPR
• CRISPR is a family of DNA sequences in bacteria and archaea.
• The sequences contain fragments of DNA from viruses that have
attacked the prokaryote.
• These fragments are used by the prokaryote to detect and destroy
DNA from similar viruses during subsequent attacks.
• These sequences play a key role in a prokaryotic defense system,
and form the basis of a technology known as CRISPR/Cas9 that
effectively and specifically changes genes within organisms.
• The CRISPR/Cas system is prokaryotic immune system that confers
resistance to foreign genetic elements such as those present within
plasmids and phages that provides a form of acquired immunity.

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Components of CRISPR-Cas system
Basically, it is composed of two components:
1. The endonuclease
The only CRISPR protein required is the Cas9 endonuclease. It has all
the components essential for:
• Binds to guide RNA: Cas9 to cut a specific genomic locus of many
possible loci. Without binding to the guide RNA, Cas9 cannot cut.
• Binds to target DNA: Cas9 endonuclease binding to the target
genomic locus is mediated both by the target sequence contained
within the gRNA and a 3-base pair sequence known as the PAM.
• Cleave target DNA resulting in a DSB: Upon target binding, Cas9
undergoes a conformational change that positions the nuclease
domains to cleave opposite strands of the target DNA. This results
in double strand break of DNA strand.
2. The synthetic guide RNA or gRNA
• In the CRISPR-Cas system, Cas9 is guided to its target sites with
the aid of two RNAs:
• crRNA which defines the genomic target for Cas9.
• tracrRNA which acts as a scaffold linking the crRNA to Cas9.
• Nowadays, these two small RNAs have been condensed into one
RNA sequence known as the guide RNA (gRNA) or single guide
RNA (sgRNA).

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Page | 62
6. Integrated methods of disease management
• No one method of control is likely to keep the crops entirely free
from virus infection.
• Therefore, different methods of disease control are applied to the
same crop based on profit or loss ratio.
• One of the main aims of integrated controls is to avoid changes in
balance of nature.

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 • In this method judicious use of chemicals in the form of insecticide
to manage vectors and in the form of weedicides to manage weeds
as alternate or collateral hosts are also used if desired.
8. Summary
• Viral coat proteins, movement proteins and replicases offer a means
of inducing virus resistance in plants using transgenic approaches.
• RNA mediated gene silencing, through formation of silencing
RNAs from double stranded viral RNAs is an effective way of
developing virus resistance in plants.
• The plant resistance genes that recognize different viral avirulence
proteins and launch a defence response against viruses or ribosomal
inactivating proteins, which are present in the cell wall and enter
cytoplasm in response to injury, can be used to develop virus
resistant transgenic.
• Some virus resistance transgenic has been commercialized for crops
like cassava, papaya, squash etc.
References
1. Edward P Rybicki. CRISPR-Cas9 strikes out in cassava transgenic
cassava expressing Cas9 is not protected from geminivirus infection.
Agricultural Biotechnology. 2019; 37:727-729.
2. Elisabeth Waigmann, Patricia Zambryski. Tobacco Mosaic Virus
Movemerit Protein-Mediated Protein Transport between Trichome
Cells. The Plant Cell. 1995; 7:2069-2079.
3. Feng Zhu, Yang-Kai Zhou, Zhao-Lin Ji, Xiao-Ren Chen. The Plant
Ribosome-Inactivating Proteins Play Important Roles in Defense against
Pathogens and Insect Pest Attacks. Frontiers in Plant science, 2018, 9.
4. Jiaying Wang, Wen Li, Junxia Cui, Xianfeng Chen. The status of
quarantine regulation on plant virus and its challenges. Journal of Plant
Science. 2020; 4:194-198.
5. Roger N Beachy. Coat-protein-mediated resistance to tobacco mosaic
virus: discovery mechanisms and exploitation. The Royal Society. 1999;
354:654-659.
6. Sergey Smirnov, Vladimir Shulaev, Nilgun E Tumer. Expression of
Pokeweed Antiviral Protein in Transgenic Plants lnduces Virus
Resistance in Grafted Wild-Type Plants independently of Salicylic Acid
Accumulation and Pathogenesis-Related Protein Synthesis. Plant
Physiology. 1997; 114:1113-1121.

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7. Sizolwenkosi Mlotshwa, Gail J Pruss, Vicki Vance. Small RNAs in viral
infection and host Defense. Trends in Plant Science. 2018; 13:375-382.
8. Tomas Canto, Peter Palukaitis. Replicase-Mediated Resistance to
Cucumber Mosaic Virus Does Not Inhibit Localization and/or
Trafficking of the Viral Movement Protein. MPMI. 1999; 12:743-747.
9. Yongsen Cao, Huanbin Zhou, Xueping Zhou and Fangfang Li. Control
of Plant Viruses by CRISPR/Cas System-Mediated Adaptive Immunity.
Frontiers in Microbiology, 2020, 11.

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Page | 66
 Chapter - 3
Biosensors: A Tool for Detection of Plant
Pathogens

Authors
Aradhna Sagwal
Ph.D. Research Scholar, Department of Plant Pathology,
College of Agriculture, CCS Haryana Agricultural University,
Hisar, Haryana, India
Promil Kapoor
Assistant Scientist, Department of Plant Pathology, CCS HAU,
Hisar, Haryana, India
Satish Kumar
Assistant Scientist, Department of Plant Pathology, CCS HAU,
Hisar, Haryana, India
Abhishek Kumar
Department of Plant Pathology, College of Agriculture, CCS
Haryana Agricultural University, Hisar, Haryana, India

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Page | 68
 Chapter - 3
Biosensors: A Tool for Detection of Plant Pathogens
Aradhna Sagwal, Promil Kapoor, Satish Kumar and Abhishek Kumar

Abstract
Food losses due to pathogen infections in crops are persistent issues in
agriculture for centuries across the globe. In order to minimize the disease
induced damage in crops, as well as to maximize productivity and ensure
agricultural sustainability, advanced disease detection and prevention in
crops are imperative. Current immunological techniques and DNA-based
techniques have been proposed for pathogen identification and detection.
However these methodologies are time-consuming and require complex
instruments. So, they are 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. They provide real time detection which makes them suitable for on-
field testing and early detection of pathogens. Receptor based sensors
include enzyme, DNA enzymatic and cell-based biosensor. Furthermore,
compared to the antibody-based sensor, bacteriophage-based sensors are
more thermostable and have longer shelf life. Nanotechnology, nano
particles and quantum dots (QDs) have also emerged as essential tools for
fast detection of a particular biological marker with extreme accuracy and
sensitivity. In addition to sensitivity, simplicity, fast processing power, cost
effectiveness, Modern nanofabrication techniques resulted in advancement
of highly sensitive biosensor. Biosensors and bioelectronics have been used
in a lot of areas of healthcare, environmental, life science research, food &
military applications. Hence, approach for early detection of pathogens with
the help of biosensors so that diseases can be managed early and cause less
yield losses require attention under the current era.
Keywords: biosensors, detection, diseases, pathogens
1. Introduction
Global food security as determined by the balance of global food
production and demand has become an important international issue in

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recent years (Keinan & Clark, 2012). Worldwide, around 800 million people
are facing the problem of malnutrition (FAO, 2017). The reason being is less
food supply and loss in the productivity. Food losses due to crop infections
from pathogens such as fungi, bacteria, viruses and nematodes are persistent
issues in agriculture for centuries across the globe (Fang & Ramasamy.,
2015). Damage caused by pests and pathogens are 14% and 13%
respectively. In order to minimize the damage in crops due to diseases
during growth, harvest and postharvest processing, as well as to maximize
productivity and to ensure agricultural sustainability, early disease detection
and prevention in crops are necessary. This figure represents the different
traditional and innovative detection methods.

(Martinelli et al., 2014)

Many direct and indirect disease identification methods are used in
agriculture now a days. A direct disease detection method includes
molecular and serological methods that are used when large numbers of
samples need to be analyzed. In these methods, the pathogens that cause
diseases such as fungi, bacteria and viruses could be directly detected to
provide accurate identification of the disease/pathogen. Laboratory-based
techniques such as polymerase chain reaction (PCR), immunofluorescence
(IF), fluorescence in-situ hybridization (FISH), enzyme-linked
immunosorbent assay (ELISA), flow cytometry (FCM) and gas
chromatography-mass spectrometry (GC-MS) are some of the direct disease
detection methods on the other hand, indirect methods identify the plant

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diseases through different parameters such as morphological change,
temperature change, transpiration rate change and volatile organic
compounds that are released by infected plants. Thermography,
hyperspectral techniques and fluorescence imaging are some of the indirect
methods.
Although these methods have been developed for plant disease
identification, but their application is limited due to multiple reasons: They
are either time consuming and destructive, demand a skilled technician,
require laboratory set-up, do not provide real-time monitoring (e.g., FISH,
ELISA, IF, FCM, GC-MS) and display low specificity e.g., imaging
techniques. So, there is need of detection methods that identify pathogen
infections in crops in a rapid, real-time and non-destructive fashion so that
timely intervention and preventive treatments can be performed to minimize
the losses in crop.
2. Detection of plant diseases using portable sensors
A wide variety of sensors have been developed and the work is going on
for developing new biosensing systems for early detection of plant diseases
with high sensitivity, stability and specificity They provide real time
detection which makes them suitable for on-field testing and early detection
of pathogen even at asymptotic stage also.
2.1 Biosensors
Biosensors are an analytical device, used for the detection of an analyte,
that combines a biological component with a physicochemical detector. It
uses specific biochemical reactions to detect chemical & biological
compounds in biological samples. It converts a biological signal into
electrical signal. International Union of Pure and Applied Chemistry
(IUPAC, 1992) defines biosensor as a “device that uses specific biochemical
reactions mediated by isolated enzymes, immuno-systems, tissues,
organelles or whole cells to detect chemical compounds usually by electrical,
thermal or optical signals”. A biosensor detects chemical and biological
compounds in a living environment with the help of a specific biological
recognition element whose property changes upon binding of the compound.
2.2 History
The first 'true' biosensor was developed by Leland C. Clark, Jr in 1956
for oxygen detection. He first established the concept of utilizing a biological
sensing element for the detection of different analytes. He is known as the
'father of biosensors' and his invention of the oxygen electrode bears his

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name: 'Clark electrode'. Enzyme electrode using immobilized glucose
oxidase enzyme was developed in 1960. In 1969, G.G. Guilbaut and J.
Montalvo developed potentiometric biosensor urease immobilized on an
ammonia electrode to detect urea. Eventually in 1975 the first commercial
biosensor was developed by Yellow Spring Instruments (YSI).
2.3 Advantages of biosensors over other methods
They are having many advantages over other methods as they are highly
Specific, highly sensitive, highly stable, biocompatible, easy to use, durable,
require only small sample volume, short analysis time, accurate, less
expensive and Detection Limit-upto Pico gram (pg).
2.4 Characteristics of biosensor
There are some basic characteristics of a biosensor
1) Linearity: Maximum linear value of the sensor calibration curve.
Linearity of the sensor must be high for the detection of high
substrate concentration.
2) Sensitivity: The value of the electrode response per substrate
concentration.
3) Selectivity: Interference of chemicals must be minimised for
obtaining the correct result.
4) Response time: The necessary time for having 95% of the
response.
2.5 Components of biosensor
Transducer: A transducer is more generally defined as a device which
converts energy from one form to another which is a combination of
Bioreceptor:- The sensitive biological element that recognizes the analyte
under study.
Electrical interface: The detector element (works in a physicochemical
way; optical, piezoelectric, electrochemical, etc.) that transforms the signal
resulting from the interaction of the analyte with the biological element into
electrical signal form.
Electronics system: Combination of electronic devices i.e. amplifier,
signal processer and display device that are primarily responsible for the
display of the results in a user-friendly way.

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2.6 Principle of biosensor

The interaction of the analyte (a) with the bioreceptor, which identifies
the stimulus (b) is designed to produce an effect measured by the transducer
(c), which converts it to an electrical signal. The output from the transducer
is amplified (d), processed (e) and displayed (f).
The principle of detection is the specific binding of the analyte of
interest to the complementary biorecognition element immobilised on a
suitable support medium. The specific interaction results in a change in one
or more physico-chemical properties (pH change, electron transfer, mass
change, heat transfer, uptake or release of gases or specific ions) which are
detected and measured by the transducer.
2.7 Methods of transduction
Electrochemical method: Chemical reactions between immobilized
biomolecule and target analyte leads to the production of some electrons or
ions and electrochemical method has the tendency to detect the changes in
electrical properties of the solution which further can be used as measuring
parameter.
They are of 3 types:
1. Amperometric biosensors use electric current signal generating
from the specific binding effect (Palchetti & Mascini., 2008) and
they are highly sensitive and detect electro-active species present in
biological test samples.
2. Potentiometric biosensors detect analyte by change in voltage signal
(Leonard et al., 2003) that is generated across an ion-selective
membrane separating two solutions.
3. Impedimetric biosensors detect analyte by change in impedance
when antibody binds with analyte.

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 4. The conductometric biosensors detect the electrical signal through a
conductive polymer, such as polyacetylene, polypyrrole or
polyaniline that is generated from the biological signal. Production
of ions or electrons in the biochemical reaction changes the overall
conductivity or resistivity of the solution that is detected by
conductometric biosensors. (Sadanandom & Napier., 2010).
Optical method: Detect changes in transmission of light. Example-
SPR-based sensors can detect the change in refractive index on the
attachment of the analyte to the metal surface (e.g., gold), that is modified
with the recognition element of a conjugated ligand (Zeng et al., 2013). In
SPR based biosensors, antibodies were initially immobilized on the thin gold
film just over the reflecting surface of waveguide. At certain wavelengths
and near IR region, strong resonance was generated in the metal when light
interact with the electron cloud. Binding of pathogen on the metal surface
results in shift in resonance to longer wavelengths and amount of shift is
proportional to the concentration of bound pathogen or toxin.
Thermistor method: Heat released or absorbed by the reactions used as
a parameter for detection.
Piezoelectric method: In Piezoelectric method, there is Coupling of the
bioelement with a piezoelectric component, (quartz-crystal) coated with gold
electrodes and Crystals made to vibrate at a specific frequency with an
electrical signal. The oscillation frequency is the electrical frequency applied
to the crystal as well as the crystal’s mass. As mass increases on binding of
molecules, the oscillation frequency of the crystal is changed and the
resulting change can be measured electrically. There will be change in the
mass of biological components as result of reaction which is measured by the
piezoelectric transducer. Example- A QCM-based sensor detects a mass
variation per unit area of the QCM crystal by measuring the change in
oscillation frequency of a quartz crystal resonator. The QCM crystal is
typically modified with a recognition element (e.g., antibodies) (Eun et al.,
2002).
Immobilization methods: With relation to biosensors, the most
superior step is the immobilization of the biological element onto the
transducer surface. Many strategies are adopted for biological elements
immobilization, e.g. binding to resin, crosslinking and encapsulation. A
high-quality immobilization must assemble the subsequent necessities:
1) Decent and rapid.

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 2) No percolation of immobilized element from the substrate and
biological elements must retain their identity after immobilization
(S.K. Sharma et al., 2003).
So, to make a viable biosensor, the biological component has to be
properly attached to the transducer. The different methods are adsorption,
covalent binding, cross linking, encapsulation and matrix entrapment.
Adsorption: It is a physical method where many substances (charcoal,
clay, cellulose, kaolin, silica gel, carbon pellets) adsorb enzymes on their
surfaces. It has some advantages as no reagents required and there is less
disruption to the enzymes and some disadvantages as attachment is weak and
suitable for short-term investigations. So, it is simplest but the main
drawback is percolation due to which this technique has short lifetime.
Covalent binding: It is most common method and this bonding has
longer life span in comparison to adsorption because of stronger bond
formation between the sensing molecule and the solid support. It uses
Glutaraldehyde, Carbodiimide, Succinimide esters, Maleinimides and
eliminates problems such as instability, diffusion and aggregation or
inactivation of biomolecules. But, the factors like pH, ionic strength etc.
cannot approach the covalent bonds and the process is complicated and time
consuming, and may involve hazardous chemicals.
Cross linking: It is a chemical fixation and it uses Glutaraldehyde,
Hexamethylene di-isocyanate to bind the biomaterial to solid supports but
having some disadvantages.
Encapsulation: It is a physical method and Biomaterial held in place
behind an inert membrane (close attachment). Membranes used are cellulose
acetate, polyurethane, PTFE collagen, polycarbonate. This process is the
blend of the advantages and eliminates the anomalies of the above two
methods: covalent bonding and adsorption. Encapsulation can provide
sufficient life span and less percolation than covalent bonding. But having
some disadvantage as encapsulated biomaterial is very susceptible to
environmental changes.
Matrix-entrapment: it is a physical method and there is physical
enclosure of biomolecule in a small space, Matrices used are chemical
polymers such as calcium alginate, polyacrylamide and sol-gel. It has a
disadvantage of creation of large barrier.
2.8 Application of biosensor
It has its wide applications in many fields of science such as:

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1. In food processing, monitoring, food authenticity, quality and safety
In food processing industry there is necessity of maintenance of quality
food products and safety processing. Example- Enzymatic biosensors reveal
a good capability to monitor the ageing of beer during storage (Ghasemi-
Varnamkhasti et al., 2012). They are also used for the detection of pathogens
in food. Presence of Escherichia coli in vegetables, is a bioindicator of faecal
contamination in food. (Arora et al., 2011). E. coli has also been measured
by detecting variation in pH caused by the ammonia (produced by urease-E.
coli antibody conjugate) using potentiometric alternating biosensing systems.
Enzymatic biosensors are used in the dairy industry as these automated flow-
based biosensors could quantify the organophosphate pesticides in milk.
2. In health care
Mycobacterium tuberculosis causing Tuberculosis, Vibrio cholerae
causing Cholera, Treponema pallidium causing Syphilis etc. are detected by
the use of biosensor.
3. In food and waterborne MO’S
E. coli causing many diseases like urinary tract infections, diarrhea etc.,
Salmonella causing Salmonellosis, Staphylococcus causing impetigo, food
poisoning, cellulitis etc. and Campylobacter are detected by the use of
biosensor.
4. In defence
Bacillus anthracis and Y. pestis are also detected by biosensor.
5. In agriculture
Many plant pathogens, pesticides and alfatoxins in food samples are
detected by different biosensors.
Other applications include:
 Measurement of Metabolites.
 Screening for sickness.
 Insulin treatment.
 Clinical psychotherapy & diagnosis of disease.
 In Military.
 Agricultural and Veterinary applications.
 Drug improvement, offense detection.
 Ecological pollution control.

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2.9 Classification

Antibody-based biosensors
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.g., pathogen of interest) to a signal that can be analyzed. Most
antibody-based biosensor uses one of the following types of electrochemical
transducers: amperometric, potentiometric, impedimetric and
conductometric. The specific combination of analyte and immobilized
antibody produces a physicochemical change, such as temperature, mass,
optical property or electrical potential. The change can be translated into a
measurable signal for detection of pathogen. In the antibody-based
biosensors different bonds like hydrophobic, ionic and hydrogen bonds are
involved in the stabilization of antigen-antibody complex.
Advantages
1) Antibody-based biosensor provides rapid and sensitive detection of
a range of pathogens.
2) The antibody-based biosensors have several advantages such as fast
detection, high sensitivity, real-time analysis and potential for
quantification.
3) These biosensors hold great value for agricultural plant pathogen
detection (Skottrup et al., 2008).
4) 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 (Perdikaris et al, 2011).

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2.9.1 Virus detection by immunosensor
QCM immunosensor Orchid viruses Torrence et al., 2006
QCM immunosensor Tobacco mosaic virus Candresse et al., 2007
SPR immunosensor Cowpea mosaic virus
SPR immunosensor Lettuce mosaic virus
SPR immunosensor Maize chlorotic mottle virus Zeng et al., 2013
Cymbidium mosaic virus
Fibre optic particle plasmon
(CymMV) or Odontoglossum Lin et al., 2014
resonance immunosensor
ringspot virus (ORSV)

2.9.2 Fungus detection by immunosensor

Biosensor Pathogen Year
SPR immunoassay Phytophthora infestans Skottrup et al., 2007
SPR immunosensor Tilletia indica Singh et al., 2010
Immuno-Impedimetric
Sclerotina sclerotium Shoute et al., 2018
biosensor
SPR immunosensor Pseudocercospora fijiensis (Luna-Moreno et al., 2019)

Other examples: Antibody-based biosensors are also used for the

detection of plant pathogens such as Fusarium culmorum (Zezza et al.,
2006), Puccinia striiformis (Skottrup et al., 2007) and Aspergillus niger
(Nugaeva et al., 2007).
In recent years, antibody-based biosensor technology has been seen on
progress upon implementation of nanotechnology-based sensor fabrication.
Limitations of antibody-based biosensors
1) As many biosensors based on antibodies focus on specific binding
with a particular antigen, issues such as the exposure of a bacterial
strain to the unfavourable conditions like environmental stress (pH
and temperature), could cause errors in the measurement.
2) More importantly, antibodies are vulnerable and are easy to get
denatured, which requires specific environment (pH, temperature,
etc.) for storage, otherwise, the behavior of antibody-based sensor
will also be compromised due to the deterioration of antibody over
time (Byrne et al., 2009).

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 This represents antibody based and DNA based methods for analyte
detection. (Fang & Ramasamy., 2015).
2.9.3 DNA/RNA-Based affinity biosensor
A recently developed new type of affinity biosensor uses nucleic acid
fragments as elements for pathogen detection. In this, Immobilization of a
ssDNA probe onto a electrode surface takes place which is able to recognize
its complementary DNA target.
Applications
1) Due to the possibility of detection at a molecular level, the DNA-
based biosensor enables early detection of diseases before any
visual symptoms appear.
2) The application of specific DNA sequences has been widely used
for detection of bacteria, fungi and genetically modified organisms.
3) Based on the specific nucleic acid hybridization of the immobilized
DNA probe on the sensor and the analyte DNA sequence, DNA-
based biosensor provides rapid, simple and economical testing of
genetic and infectious diseases.
The most commonly adopted DNA probe is single stranded DNA
(ssDNA) on electrodes with electroactive indicators to measure hybridization
between probe DNA and the complementary DNA analyte (Eun et al.,
2000).
Types of-DNA-based biosensors
Depending on their mode of transduction: Different types biosensors
are optical, piezoelectric, strip type and electrochemical DNA biosensors.

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 Optical DNA biosensors transduce the emission signal of a fluorescent
label. The detection of DNA analyte is accomplished through a variation in
physio-chemical properties such as temperature, mass, optical property and
electrical property as a result of double-stranded DNA (dsDNA)
hybridization occurs during the analyte recognition. The optical DNA-based
biosensors are further classified into three types-molecular beacons (MB),
surface plasmon resonance (SPR) and quantum-dots.
Piezoelectric DNA biosensors detect the analyte by using a quartz
crystal that oscillates at a specific frequency at an applied oscillating voltage.
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 (Eun & Wong., 2000).
Advantages
1) Bacterial pathogens are easily detected by these biosensors due to
their unique nucleic acid sequence, which can be specifically
hybridized with the complementary DNA probe. The recognition of
analyte DNA depends upon the formation of stable hydrogen bonds
between the DNA probe and analyte DNA sequence.
2) In addition to DNA-DNA hybridization for bacterial detection, the
specific hybridization of DNA and complementary RNA was used
for the detection of plant viruses by molecular beacons and QCM
techniques. Example-CymMV and ORSV were detected by QCM
DNA-based biosensor with a designed DNA probe modified with a
mercaptohexyl group. The limits of detection of CymMV and
ORSV were as low as approximately 1 ng in purified RNA and 10
ng in the crude sap (Eun et al., 2002).
3) Although the application of DNA-based biosensors for plant disease
detection is promising, step of PCR have to be performed prior to
the probing process as small quantity of nucleic acid present in the
bacteria cells (Ivnitski et al., 2000).
Other examples
DNA Biosensor Pathogen Year
Cymbidium mosaic virus (CymMV) and Valladares et
DNA based biosensor
Odontoglossum ringspot virus (ORSV) al., 2012
SPR based DNA Fusarium culmorum Zezza et al.,

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 biosensor 2020
Liquid crystal-based Mashooq khan
Erwinia carotovora and Rhizoctonia solani
DNA biosensor et al., 2016

Limitations of DNA based biosensors-include the requirement for the

synthesis of specific DNA probe, amplification of DNA, high cost (DNA-
based molecular beacons) and unsuitability for real-time detection (DNA-
based piezoelectric biosensor).
Enzymatic electrochemical biosensors
The use of enzyme as bio-recognition element provides highly specific
detection of the target analyte because of the high specificity of the 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 leads to the formation of electrical signal
(current) which, 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.
The enzymatic electrochemical biosensors are not successfully
commercialized, unlike other types of biosensors. For plant pathogen
detection, enzymatic biosensors could be used if the target VOC could be
collected in the form of a liquid sample. It has been studied 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 (Thomas et al., 1999). Many of the
VOCs produced by infected crops are alcohols and aldehydes such as cis-3-
hexen-1-ol and trans-2-hexanal, which are catalyzed by alcohol
dehydrogenase enzymes. Correspondingly, 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. In addition to those specific
volatile organic compounds, the common phytohormones such as auxin,
cytokinins and gibberellins which are indicative of plant health that could
also be deactivated by oxidases. Gibberellin is deactivated by GA-2-oxidases
which provides the potential for fabrication of gibberellin detection for plant
disease prediction (Kulagina et al., 1999).

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 This table represents VOCs emitted from whole, intact tomato plants or
detached leaves, and biotic stress causing agents responsible for increase in
VOC emissions (Jansen et al., 2009).
Although enzyme-based biosensors usually provide high sensitivity and
specificity for the detection, stability of enzymes is of major problem. In
addition, the enzyme catalysis varies with factors such as temperature and
pH which could disturb the accuracy of the biosensor.
Bacteriophage based biosensor
Bacteriophage is a virus, composed of protein capsid that encapsulates
DNA or RNA genome. 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 (McGrath & Vansinderen, 2007).
In addition to phage therapy, bacteriophage is also emerging as a
promising alternative for pathogen detection due to high selectivity,
sensitivity, low cost and higher thermostability (Kretzer et al., 2000) (Brigati
et al, 2005) (Neufeld et al., 2003). Upon the interaction between
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
(Adriaenssens et al., 2012) and tomatoes (Fujiwara et al., 2011). A Phage-
based diagnostic assay used for detecting and identifying Pseudomonas
cannabina pv. Alisalensis from different cultures and diseased plant samples.
The bacterial luxAB reporter genes (encoding the luciferase) integrated into
P. cannabina pv Alisalensis phage PBSPCA1. In the presence of target
pathogen cell, the lux AB are expressed in the host cell on specific infection
through the phage. The expressed luciferase results in the light emission in
presence of n-decanal, oxygen (Schofield et al., 2013).

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 (Schofield et al., 2013)
In addition to P. cannabina pv. alisalensi, bacteriophages that
specifically bind with other pathogens are also discovered such as
Pseudomonas syringae pv. Actinidiae, which causes bacterial canker of
kiwifruits, and Ralstonia solanacearum, a soil borne bacterium that is the
causative agent of bacterial wilt in many important crops (Frampton et al.,
2014) (Askora et al., 2009) The progress in discovering more bacteriophages
provides the possibility for fabrication of many more bacteriophage-based
sensors for plant disease detection.
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 having longer shelf life. Bacteriophage-based biosensors are also
capable of differentiating between the live and dead bacterial pathogens
which reduces the false positive signals during measurement.
Disadvantages-Phage-based biosensors take a longer time for measuring
the pathogen because of the complex sample preparation requirement (Tlili
et al., 2013). Bacteriophage-based sensor can only be fabricated for detection
of bacteria rather than fungi and viruses which severely limits its application
for the majority of crops that are affected by fungal pathogens.

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2.9.4 Nanotechnology and nanomaterials
Biosensor Platform Based on Nanomaterials:
Nanotechnology display the preparation of various nanoparticles and
nanostructures. Nanoparticles provides fascinating electronic and optical
properties and can be synthesized using different types of materials for
electronics and sensing applications (Shipway et al., 2000). For biosensing
application, the limit of detection and the overall performance of a biosensor
can be greatly improved by using different nanomaterials for their
construction as transducers. The popularity of nanomaterials for sensor
development due to to the friendly platform due to reason, it provides the
assembly of bio-recognition element, the high surface area, high electronic
conductivity and plasmonic properties of nanomaterials that improve the
limit of detection. 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.
In short, Nanotechnology, nano particles and quantum dots (QDs)
emerged as essential tools for fast detection of a particular biological marker
with high accuracy and sensitivity (Mohammad Khiyami et al., 2014).
Examples-Nanoparticles used with other biological materials such as
antibody for detecting Xanthomonas axonopodis that causes bacterial spot
disease (Yao et al., 2009). Gold nanoparticle-based optical immunosensors
have been developed for detection of Karnal bunt disease in wheat using
surface plasmon resonance (SPR) (Singh et al., 2010). 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 causes bacterial spot diseases
in Solanaceae plant (Yao et al., 2009).
Quantum dots (QD) have also been used for biosensor construction for
disease detection (Frasco & Chaniotakis., 2009). Due to their unique and
advantageous optical properties, they also used for disease detection using
fluorescence resonance energy transfer (FRET) mechanism (Algar et al.
2008), which describes energy transfer between two light-reactive
molecules.
QD-FRET-based sensors have been developed for
Phytoplasma disease detection like witches’ broom disease of lime
(WBDL) caused by Candidatus phytoplasma aurantifolia. The
immunosensor constructed showed a high sensitivity and specificity of 100%
and a detection limit of 5 ca. P. aurantifolia/µL (Rad et al., 2012).
Additionally, QD-FRET was also reported to detect disease vectors.

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 Rhizomania, which is considered the most destructive disease in sugar
beet, is caused by beet necrotic yellow vein virus (BNYVV). Polymyxa betae
(Keskin), the only known vector of BNYVV, for transmission of the virus to
the plants was successfully proved to be detected by the QD-FRET-based
sensor (Safarpour et al., 2012).
Gold nanoparticles are widely used nanomaterials due to their high
electroactivity and electronic conductivity for electron transfer (Mandler &
Kraus-ophir., 2011). Gold nanoparticles used for sensitive
immunochromatographic diagnostics of potato brown rot (Razo et al., 2019).
Recently, nanomaterial-based electrochemical sensors have been reported for
plant disease detection (Umasankar & Ramasamy., 2013). The application of
gold nanoparticle (AuNP) modified electrode was also found for the
electrochemical detection of methyl salicylate, a key plant volatile organic
compound released by plants during infections. However, in addition to gold
nanoparticles, semiconductive metal oxide nanoparticles also found for VOC
detection as they have many advantages such as low cost and suitability for
electron conduction for amperometric signal. It has been found that
application of metal oxide nanoparticles such as SnO2 and TiO2 for VOC
detection such as p-ethylguaiacol produced by infected strawberry (Fang et
al., 2014). The limit of detection achieved was in nanomolar concentration
range. In addition to the detection of plant VOC which is indicative of a
particular disease, nanoparticles are also be used for detection of many
compounds released by the pathogen itself. Different types of
phytopathogens-phytobacteria, viruses and fungi-are also detected through
nanoparticle based amperometric biosensors (Boonham et al., 2008)
(Chartuprayoon et al. 2010).

(Fang and Ramasamy., 2015)

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Conclusion
Biosensors provide real time monitoring and early detection of
pathogens so that diseases can be managed early and cause less yield losses.
Biosensors and bioelectronics have been used in a lot of areas of healthcare,
environmental, life science research, food & military applications. In
addition to sensitivity, simplicity, fast processing power, cost effectiveness,
many modern nanofabrication techniques resulted in advancement of highly
sensitive biosensor.
Future prospects
Due to its wide applications, there is need to bring this technology to
make it commercially available for ease. With exposure to the commercial
market, the applications of the technology would be greatly enhanced.
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 Chapter - 4
RNA Interference: A Recent Approach for Plant
Diseases Management

Authors
Sunil Kumar
Ph.D. Scholars, Department of Plant Pathology, SKNCOA,
Jobner Sri Karan Narendra Agriculture University, Jobner,
Jaipur, Rajasthan, India
Shivam Maurya
Ph.D. Scholars, Department of Plant Pathology, SKNCOA,
Jobner Sri Karan Narendra Agriculture University, Jobner,
Jaipur, Rajasthan, India
Stayadev Prajapati
Ph.D. Scholars, Department of Plant Pathology, SKNCOA,
Jobner Sri Karan Narendra Agriculture University, Jobner,
Jaipur, Rajasthan, India
Naresh Kumar
Ph.D. Scholars, Department of Plant Pathology, SKNCOA,
Jobner Sri Karan Narendra Agriculture University, Jobner,
Jaipur, Rajasthan, India

Page | 91
Page | 92
 Chapter - 4
RNA Interference: A Recent Approach for Plant Diseases
Management
Sunil Kumar, Shivam Maurya, Stayadev Prajapati and Naresh Kumar

Abstract
Plant diseases represent a tremendous risk to crop production globally.
Varieties in their genomes cause choice to support the individuals who can
endure pesticides and Bacillus thuringiensis (Bt) crops. Despite the fact that
plant rearing has been the old-style methods for controlling the plant genome
to create safe cultivar for controlling plant diseases, the coming of hereditary
building gives a completely new methodology being sought after to render
plants impervious to parasites, microbes, infections and nematodes. RNA
obstruction (RNAi) innovation has risen to be a promising restorative tool to
moderate the intrinsic dangers, for example, the utilization of a particular
transgene, marker quality or quality control groupings related with
advancement of customary transgenics. Quieting explicit qualities by RNAi
is an alluring regular answer for this issue as illness safe transgenic plants
can be created inside an administrative system. Ongoing examinations have
been fruitful in delivering powerful hushing impacts by utilizing objective
twofold stranded RNAs through a compelling vector framework. Transgenic
plants communicating RNAi vectors, just as, dsRNA containing crop
splashes have been fruitful for productive control of plant pathogens
influencing financially significant harvest species.
Keywords: RNA interference, transgene, management, plant disease
Introduction
The process of RNAi was referred to as "co-suppression" and "quelling"
when observed prior to the knowledge of an RNA-related mechanism. The
discovery of RNAi was preceded first by observations of transcriptional
inhibition by antisense RNA expressed in transgenic plants and more directly
by reports of unexpected outcomes in experiments performed by plant
scientists in the United States and the Netherlands in the early 1990s. In an
attempt to alter flower colors in petunias, researchers introduced additional

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copies of a gene encoding chalcone synthase, a key enzyme for flower
pigmentation into petunia plants of normally pink or violet flower color. The
overexpressed gene was expected to result in darker flowers, but instead
caused some flowers to have less visible purple pigment, sometimes in
variegated patterns, indicating that the activity of chalcone synthase had
been substantially decreased or became suppressed in a context-specific
manner. This would later be explained as the result of the transgene being
inserted adjacent to promoters in the opposite direction in various positions
throughout the genomes of some transformants, thus leading to expression of
antisense transcripts and gene silencing when these promoters are active.
Another early observation of RNAi came from a study of the fungus
Neurospora crassa although it was not immediately recognized as related.
Further investigation of the phenomenon in plants indicated that the down
regulation was due to post-transcriptional inhibition of gene expression via
an increased rate of mRNA degradation. This phenomenon was called co-
suppression of gene expression, but the molecular mechanism remained
unknown. Not long after, plant virologists working on improving plant
resistance to viral diseases observed a similar unexpected phenomenon.
While it was known that plants expressing virus-specific proteins showed
enhanced tolerance or resistance to viral infection, it was not expected that
plants carrying only short, non-coding regions of viral RNA sequences
would show similar levels of protection. Researchers believed that viral
RNA produced by transgenes could also inhibit viral replication. The reverse
experiment, in which short sequences of plant genes were introduced into
viruses, showed that the targeted gene was suppressed in an infected plant.
This phenomenon was labeled "virus-induced gene silencing" (VIGS), and
the set of such phenomena were collectively called post transcriptional gene
silencing. After these initial observations in plants, laboratories searched for
this phenomenon in other organisms. Craig C. Mello and Andrew Fire's 1998
Nature paper reported a potent gene silencing effect after injecting double
stranded RNA into C. elegans. In investigating the regulation of muscle
protein production, they observed that neither mRNA nor antisense
RNA injections had an effect on protein production, but double-stranded
RNA successfully silenced the targeted gene. As a result of this work, they
coined the term RNAi. This discovery represented the first identification of
the causative agent for the phenomenon. Fire and Mello were awarded the
2006 Nobel Prize in Physiology or Medicine. To offset the crop losses from
pathogens, various attempts have been made in the field of disease
management since inception of green revolution. Pesticides have been
traditionally used on crop to prevent crop damages. Once pesticides were
discovered to pollute the environment and be harmful to human health,

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agriculture research began to focus on alternative safer methods. In last two
and half decades, much attention has been paid on integrated disease
management practices which make disease control inexpensive and safe.
Plant breeding has been the classical means of manipulating the plant
genome to develop resistant cultivar for controlling plant diseases. Further
study of genetic host resistance fulfils this requirement but is a continuous
endeavor as the boom and bust cycle goes on in the process of co-evolution,
though therapeutic tools based on current molecular biology hold the key
after the exploitation of traditional breeding and biotechnological methods
likely use molecular markers for identification, mapping, cloning of pest and
disease resistant genes, and their utilization by introgression, pyramiding,
and development of transgenics. The inherent risks associated with
traditional transgenics can be mitigated by new and innovative strategies,
and transgenic plants can be produced within a regulatory framework. RNA
interference is a natural process, which silences specific genes before being
translated. RNAi inducers, in the form of transgenic plants or a crop spray,
have the potential to effectively silence specific genes. During the last
decade our knowledge repertoire of RNA-mediated functions has hugely
increased with the discovery of small non-coding RNAs, which play a
central part in a process called RNA silencing. Ironically the very important
phenomenon of co-suppression has recently been recognized as a
manifestation of RNA interference (RNAi), an endogenous pathway for
negative posttranscriptional regulation. RNAi has revolutionized the
possibilities for creating custom “knock-downs” of gene activity. RNAi
operates in both plants and animals and uses double stranded (dsRNA) as a
trigger that targets homologous mRNAs for degradation or inhibiting its
transcription or translation whereby susceptible genes can be silenced. This
RNA-mediated gene control technology has provided new platforms for
developing eco-friendly molecular tools for crop improvement by
suppressing the genes responsible for various stresses and improving novel
traits in plants including disease resistance. Also, it will be a promising
future therapeutic agent to combat plant invaders. With this technology it is
possible to silence a gene throughout an organism or in specific tissues,
offering the versatility to partially silence or completely turn off genes in
both cultured cells and whole organisms, and can selectively silence genes at
particular stages of the organism’s life cycle. Transgenic plants would be
cost effective by producing RNAi inducers throughout a plant’s life
constantly silencing different pathogen genes. The applications of this
technology in the improvement of plants with special reference to disease
management are discussed below.

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Virus induced gene silencing (VIGS)
Virus-induced gene silencing (VIGS) is a technology that exploits an
RNA-mediated antiviral defense mechanism. In plants infected with
unmodified viruses the mechanism is specifically targeted against the viral
genome. However, with virus vectors carrying inserts derived from host
genes the process can be additionally targeted against the corresponding
mRNAs. VIGS has been used widely in plants for analysis of gene function
and has been adapted for high-throughput functional genomics. Until now
most applications of VIGS have been in Nicotiana benthamiana. However,
new vector systems and methods are being developed that could be used in
other plants, including Arabidopsis.

Fig 1: General Mechanism of VIGS

Most viruses are plus-strand RNA viruses or satellites, whereas, the
tomato golden mosaic virus (TGMV) and the cabbage leaf curl virus
(CaLCuV) are DNA viruses. Though RNA viruses replicate in the
cytoplasm, DNA viruses replicate in plant nuclei using the host DNA
replication machinery. Both types of viruses induce diffusible, homology-
dependent systemic silencing of endogenous genes. However, the extent of

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silencing spread and the severity of viral symptoms can vary significantly in
different host plants and host/virus combinations. With the variety of viruses
and the diversity of infection patterns, transmission vectors, and plant
defenses it is not surprising that viruses differ with respect to silencing.
Because the continuing development of virus-based silencing vectors can
extend VIGS to economically important plants, it is useful to consider some
of the characteristics of successful VIGS vectors.
RNAi Strategies in plant disease management
Despite substantial advances in plant disease management strategies, our
global food supply is still threatened by a multitude of pathogens and pests.
This changed scenario warrants us to respond more efficiently and
effectively to this problem. The situation demands judicious blending of
conventional, unconventional and frontier technologies. In this sense, RNAi

Fig 2: General Mechanism of RNA interference

technology has emerged as one of the most potential and promising
strategies for enhancing the building of resistance in plants to combat
various fungal, bacterial, viral and nematode diseases causing huge losses in
important agricultural crops. The nature of this biological phenomenon has
been evaluated in a number of host-pathogen systems and effectively used to
silence the action of pathogens. Many of the examples listed below illustrate
the possibilities for commercial exploitation of this inherent biological
mechanism to generate disease-resistant plants in the future by taking
advantage of this approach.

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Management of plant pathogenic fungi
RNA-mediated gene silencing (RNA silencing) is used as a reverse tool
for gene targeting in fungi. Homology based gene silencing induced by
transgenes (cosuppression), antisense, or dsRNA has been demonstrated in
many plant pathogenic fungi, including Cladosporium fulvum, Magnaporthe
oryzae, Venturia inaequalis, Neurospora crassa, Aspergillus nidulans and
Fusarium graminearum whether it is suitable for large-scale mutagenesis in
fungal pathogens. The hypermorphic mechanism of RNA interference
implies that this technique can also be applicable to all those plant
pathogenic fungi, which are polyploid and polykaryotic in nature. HCf-1, a
gene that codes for a hydrophobin of the tomato pathogen C. fulvum was
suppressed by ectopic integration of homologous transgenes. Transformation
of Cladosporium fulvum with DNA containing a truncated copy of the
hydrophobin gene HCf-1 caused co-suppression of hydrophobin synthesis in
30% of the transformants. The suppressed isolates had a hydrophilic
phenotype, lower levels of HCf-1 mRNA than wild type and contain
multiple copies of the plasmid integrated as tandem repeats at ectopic sites in
the genome. The transcription rate of HCf-1 in the co-suppressed isolates
was higher than in the untransformed strains, suggesting that silencing acted
at the post-transcriptional level. This was due to ectopic integration of the
transgene next to promoters which initiate transcription to form antisense
RNA, and that this in turn determined the down-regulation of HCf-1. Gene
silencing was not associated with DNA cytosine methylation. The silencing
of cgl1 and cgl2 genes using the cgl2 hairpin construct in Cladosporium,
though the effect was possibly restricted to highly homologous genes (exons
of cgl 1 and cgl 2 are 87% identical). However, the less homologous cgl 3
(53% overall identity to cgl 2) was not affected as the target specificity
always depends upon the actual sequence alignment and moreover, short
regions of high density that led to unwanted off-target effects. Such a
strategy could be exploited for protecting the consumable products of
vegetables and fruit crops from the post-harvest diseases caused by different
plant pathogens in the future. Using the hairpin vector technology, have been
able to trigger simultaneous high frequency silencing of a green fluorescent
protein (GFP) transgene and an endogenous trihydroxynaphthalene reductase
gene (THN) in V. inaequalis. GFP transgene, acting as an easily detectable
visible marker while the trihydroxynaphthalene reductase gene (THN)
playing a role in melanin biosynthesis. High frequency gene silencing was
achieved using hairpin constructs for the GFP or the THN genes transferred
by Agrobacterium (71 and 61%, respectively). THN-silenced transformants
exhibited a distinctive light brown phenotype and maintained the ability to

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infect apple. Silencing of both genes with this construct occurred at a
frequency of 51% of all the transformants. Multiple gene silencing has been
achieved in Cryptococcus neoformans using chimeric hairpin constructs and
in plants using partial sense constructs. The first effort towards the
systematic silencing of Magnaporthe grisea, a causal organism of rice blast
was carried out by using the enhanced green florescent protein gene as a
model. To assess the ability of RNA species induce silencing in fungus,
plasmid construct expressing sense, antisense and hairpin RNA were
introduced into an eGFP expressing transformants. The fluorescence of
eGFP in the transformants was silenced much more efficiently by hairpin
RNA of eGFP than by other RNA species. In the silenced transformants, the
accumulation of eGFP mRNA was drastically reduced, but not methylation
of coding or promoter regions was involved.
Management of plant pathogenic bacteria
One of the striking examples of bacterial disease management where
RNAi showed a remarkable type of gene regulation was documented they
developed a crown gall disease management strategy that targets the process
of tumourogenesis (gall formation) by initiating RNAi of the iaaM and ipt
oncogenes. Expression of these genes is a prerequisite for wild type tumor
formation. Transgenic Arabidopsis thaliana and Lycopersicon esculentum
transformed with RNAi constructs, targeting iaaM and ipt gene(s) show
resistance to crown gall disease. Transgenic plants generated through this
technology contained a modified version of these two bacterial gene(s)
required to cause the disease and was the first report to manage a major
bacterial disease through RNAi. The extra genes recognize and effectively
shut down the expression of the corresponding bacterial gene during
infection, thus preventing the spread of infection. The incoming bacteria
could not make the hormones needed to cause tumors, and plants deficient in
silencing were hyper-susceptible to A. tumefaciens. Successful infection
relied on a potent anti-silencing state established in tumors whereby siRNA
synthesis is specifically inhibited. The procedure can be exploited to develop
broad-spectrum resistance in ornamental and horticultural plants which are
susceptible to crown gall tumorigenesis. This approach can be advocated for
the effective management of those pathogens which multiply very rapid, and
results in tumor formation such as Albugo candida, Synchytrium
endobioticum and Erwinia amylovora among others. The natsiRNA
(natsiRNAATGB2) was strongly induced in Arabidopsis upon infection by
Pseudomonas syringae pv. tomato and down regulates a PPRL gene that
encodes a negative regulator of the RPS2 disease resistance pathway. As a

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result, the induction of nat-siRNAATGB2 increases the RPS2mediated race-
specific resistance against P. syringae pv. tomato in Arabidopsis. Recently,
the accumulation of a new class of sRNA, 30 to 40 nucleotides in length,
termed long-siRNAs (lsiRNAs), was found associated with P. syringae
infection. One of these lsiRNAs, AtlsiRNA-1, contributes to plant bacterial
resistance by silencing AtRAP, a negative regulator of plant defense. A
Pseudomonas bacterial flagellin-derived peptide is found to induce the
accumulation of miR393 in Arabidopsis. miR393 negatively regulates
mRNAs of F-box auxin receptors, resulting in increased resistance to the
bacterium (P. syringae), and the overexpression of miR393 was shown to
reduce the plant’s bacterial titer by five-fold.
Management of plant pathogenic viruses
Antiviral RNAi technology has been used for viral disease management
in human cell lines. Such silencing mechanisms (RNAi) can also be
exploited to protect and manage viral infections in plants. The effectiveness
of the technology in generating virus resistant plants was first reported to
PVY in potato, harboring vectors for simultaneous expression of both sense
and antisense transcripts of the helper-component proteinase (HC-Pro) gene.
The P1/HC-Pro suppressors from the potyvirus inhibited silencing at a step
down stream of dsRNA processing, possibly by preventing the unwinding of
duplex siRNAs, or the incorporation into RISC or both. The utilization of
RNAi technology has resulted in inducing immunity reactions against
several other viruses in different plant-virus systems. In phyto-pathogenic
DNA viruses like geminiviruses, the non-coding intergenic region of
Mungbean yellow mosaic India virus (MYMIV) was expressed as hairpin
construct under the control of the 35S promoter, and used to holistically
inoculate MYMIV-infected black gram plants, showing a complete recovery
from infection, which lasted until senescence. RNAi-mediated silencing of
geminiviruses using transient protoplast assay where protoplasts were co-
transferred with a siRNA designed to the replicase (Rep)-coding sequence of
African cassava mosaic virus (ACMV), and the genomic DNA of ACMV,
resulted in 99% reduction of Rep transcripts and 66% reduction of viral
DNA. It was observed that siRNA was able to silence a closely related strain
of ACMV but not a more distantly related virus. More than 40 viral
suppressors have been identified in plant viruses. Virus suppressors indicated
that they interfere with systemic signaling for silencing. During the last few
years, the p69 encoded by Turnip yellow mosaic virus has been identified as
a silencing suppressor preventing host RDR dependent secondary dsRNA
synthesis. P14 protein encoded by Aureus viruses suppressed both virus and

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transgene-induced silencing by sequestering both long dsRNA and siRNA
without size specificity. Multiple suppressors have been reported in the
Citrus tristeza virus, where p20 and coat protein (CP) play important roles in
suppression of the silencing signal, and p23 inhibited intracellular silencing.
Multiple viral components, viral RNAs and putative RNA replicase proteins
were reported for the silencing or suppression of the Red clover necrotic
mosaic virus. In this case, the RNA silencing machinery deprived of DICER-
like enzymes by the viral replication complexes appeared to be the cause of
the suppression. Pns10 encoded by Rice dwarf virus suppressed local and
systemic S-PTGS but not IR-PTGS suggesting that Pns10 also targets an
upstream step of dsRNA formation in the silencing pathway.
Management of plant parasitic nematodes
Several major plant parasitic nematodes such as the root-knot
(Meloidogyne spp.) and cyst (Heterodera spp.) along with other minor
nematodes cause significant damage to important crops like legumes,
vegetables, and cereals in most parts of the world, and continue to threaten
these agricultural crops. So a natural, ecofriendly defense strategy that
delivers a cost-effective control of plant parasitic nematodes is needed but is
difficult to achieve through conventional approaches. However, the birth of
RNAi technology from classical Caenorhabditis elegans studies has shown
the ways and means to explore the possibilities of this mechanism for
protecting plants from nematode damage. In this context, two approaches
have been advocated, one of them relies on targeting plant genes that are
involved with the infection process, and the second approach targets
essential genes within the nematode. RNAi can be induced in C. elegans by
feeding it dsRNA, so it was reasoned that expressing hpRNAs-containing
sequences of vital nematode genes in the host plant might deliver dsRNA to
a feeding nematode to incapacitate or kill it. After the demonstration of gene
silencing using siRNA duplexes in the nematode the use of RNAi has rapidly
emerged as the technique of choice for plant nematologists, especially for
nematode management in agriculture. RNAi-mediated suppression of a gene
plays an indispensable role in hampering the nematode development and
may affect the progression of pathogenesis in direct or indirect ways. The
efficacy of RNAi in plant parasitic nematode management and a wide range
of genes have been targeted for silencing in cyst and root-knot nematode
species. RNAi in the context of phyto-parasitic nematodes was used as early
as the beginning of this century, when stimulation of oral ingestion by
second-stage juveniles of cyst nematodes Heterodera glycines, G. pallida
and root-knot nematode M. incognita was achieved by using octopamine.

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Later on, resorcinol- and serotonin-inducing dsRNA uptake by second stage
juvenile of M. incognita was found to be more effective than octopamine.
The genes targeted by RNAi to date are expressed in a range of different
tissues and cell types. The ingested dsRNA can silence genes in the intestine,
female reproductive system, sperm and both subcentral and dorsal
oesophageal glands. Uptake of dsRNA from the gut is a proven route to
systemic RNAi in C. elegans. The systemic nature of RNAi in plant parasitic
nematodes following ingestion of dsRNA suggests that they share similar
uptake and dispersal pathways. However, RNAi of a chitin synthase gene
expressed in the eggs of Meloidogyne artiella was achieved by soaking
intact eggs contained within their gelatinous matrix in a solution containing
dsRNA. The enzyme plays a role in the synthesis of the chitinous layer in the
eggshell. Depletion of its transcript by RNAi led to a reduction of stainable
chitin in eggshells and a delay in hatching of juveniles from treated eggs.
Similarly, RNAi targeting for cysteine proteinase transcripts did not reduce
parasitic population of established nematodes on plants but result into the
alteration of their sexual fate in favour of males at 14 days after invasion
(Urwin et al., 2002). On the other hand, H. glycines exposed to dsRNA
corresponding to a protein with homology to C-type lectins did not affect
sexual fate, but 41% fewer nematodes were recovered from the plants.
However, treatment with dsRNA corresponding to the major sperm protein
(MSP) had no effect on nematode development or sexual fate 14 days after
treatment. In addition to this, reduction in transcript abundance for targeted
mRNAs in the infective juvenile and for MSP transcripts when males
reached sexual maturity and sperm are produced. The efficient FITC uptake
by soaking M. incognita, 90 to 95% of individuals swallowed the dye when
the target was a dual oxidase (an enzyme comprised with a peroxidase
domain EF-hands and NADPH oxidase domain and potentially involved in
extracellular matrix development). The effect of RNAi was observed when
root knot nematode (RKN) juveniles were fed on dual oxidase derived
dsRNA, the reduction in the number and size of established females at 14-
and 35-days post infection with an overall reduction of 70% in egg
production. RNAi has also been induced for a chitin synthase gene that is
expressed in the eggshells of M. artiella after soaking its developing eggs in
a dsRNA. Heterodera schachtii induces syncytial feeding structures in the
roots of host plants, and this requires the up-regulation of Suc transporter
genes to facilitate increased nutrient flow to the developing structure.
Targeting these genes and down-regulating them with RNAi resulted in a
significant reduction of female nematode development. Indeed, tobacco
plants transformed with hpRNA constructs against two root-knot nematode

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genes have shown such an effect the target mRNAs in the plant parasitic
nematodes were dramatically reduced, and the plants showed effective
resistance against the parasite.
Conclusion
The field of RNAi is moving at an impressive pace and generating
exciting results associated with RNAi, transgene silencing and transposon
mobilization. This technology can be considered an eco-friendly, biosafe and
ever green technology as it eliminates even certain risks associated with
development of transgenic plants carrying first generation constructs (binary
vectors and sense and antisense genes). As witnessed from earlier strategies
for obtaining viral resistant plants, the expression of protein products from
the transgene of interest risked hetero-encapsidation through protein
interactions between target and non-target viral gene product, resulted in the
development of a non-aphid transmissible strain of Zucchini yellow mosaic
virus to aphid-transmissible strain from a transgene expressing a plum pox
capsid protein. To facilitate silence gene expression, time specific and
inducible promoters active in the target tissues which could, when required,
minimize “off-target” effects. Conventional transgenic technologies
generally need the expression of whole genes, which are in contrast to
comparatively small size of the RNAi transgene required for silencing,
permitting multiple genes to be targeted in a single construct. For changing
stages in a particular metabolic pathway or resisting multiple pathogen
attack, this would assist to lessen the amount of manipulation and time
required to accomplish the desired traits. In future opportunities, RNAi may
even hold guarantee for development of gene-specific therapeutics or a
complete understanding of genomics. With the methodical research in RNAi
mechanisms and understanding the entire development of RNAi technology,
it would be feasible to create a new biological science offering massive
economic and social spin-offs.
References
1. Dalakouras A, Wassenegger M, Dadami E, Ganopoulos I, Pappas ML,
Papadopoulou K. Genetically modified organism-free RNA
interference: exogenous application of RNA molecules in plants. Plant
Physiol. 2020; 182(1):38-50.
2. Muhammad T, Zhang F, Zhang Y, Liang Y. RNA interference: a natural
immune system of plants to counteract biotic stressors. Cells. 2019;
8(1):38.

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3. Dong OX, Ronald PC. Genetic engineering for disease resistance in
plants: recent progress and future perspectives. Plant Physiol. 2019;
180(1):26-38.
4. Gomez MA, Lin ZD, Moll T et al. Simultaneous CRISPR/Cas9-
mediated editing of cassava eIF4E isoforms nCBP-1 and nCBP-2
reduces cassava brown streak disease symptom severity and incidence.
Plant Biotechnol. J. 2019; 17(2):421-434.
5. Hudzik C, Hou Y, Ma W, Axtell MJ. Exchange of small regulatory
RNAs between plants and their pests. Plant Physiol. 2020; 182(1):51-62.
6. Baulcombe DC. VIGS, HIGS and FIGS: small RNA silencing in the
interactions of viruses or filamentous organisms with their plant hosts.
Curr. Opin. Plant Biol. 2015; 26:141-146.
7. Chen W, Kastner C, Nowara D et al. Host-induced silencing of
Fusarium culmorum genes protects wheat from infection. J Exp. Bot.
2016; 67(17):4979-4991.
8. Hajeri S, Killiny N, El-Mohtar C, Dawson WO, Gowda S. Citrus
tristeza virus-based RNAi in citrus plants induces gene silencing in
Diaphorina citri, a phloem-sap sucking insect vector of citrus greening
disease (Huanglongbing). J Biotechnol. 2014; 176:42-49.
9. Ham BK, Lucas WJ. Phloem-mobile RNAs as systemic signaling
agents. Annu. Rev. Plant Biol. 2017; 68:173-195.
10. Cai Q, Qiao L, Wang M et al. Plants send small RNAs in extracellular
vesicles to fungal pathogen to silence virulence genes. Science. 2018;
360(6393):1126-1129.
11. Hudzik C, Hou Y, Ma W, Axtell MJ. Exchange of small regulatory
RNAs between plants and their pests. Plant Physiol. 2020; 182(1):51-62.

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 Chapter - 5
Molecular Diagnostics Tools for Detection of
Plant Pathogens

Authors
Anil Kumar
Department of Plant Pathology, Dr. Rajendra Prasad Central
Agricultural University, Pusa, Bihar, India
Mohit Kumar
Department of Plant Pathology, Dr. Rajendra Prasad Central
Agricultural University, Pusa, Bihar, India
Mampi Suklabaidya
Department of Plant Pathology, Dr. Rajendra Prasad Central
Agricultural University, Pusa, Bihar, India
Rahul Patidar
Department of Plant Pathology, Dr. Rajendra Prasad Central
Agricultural University, Pusa, Bihar, India

Page | 105
Page | 106
 Chapter - 5
Molecular Diagnostics Tools for Detection of Plant
Pathogens
Anil Kumar, Mohit Kumar, Mampi Suklabaidya and Rahul Patidar

Abstract
The molecular techniques are the most important tools for quick, fast,
and accurate detection or identification of plant pathogenic fungi, bacteria,
virus, and viroid based on their genetic material, DNA, RNA, gene, and size
of nucleotides using different techniques such AFLPs, RAPD, PAGE,
Polyprobes, multiplexing potential of next-generation sequencing, probe
mix, LAMP, NASBA, FISH, Co-PCR, Multiplex PCR, Real-time
quantitative PCR and Biosensors for proper diagnosis of different plant
diseases and their causal organism to timely management of plant pathogens
using by suitable control measures that are cultural practices, physical
methods, use of biocontrol agents and application of chemicals and it has
been reduced economic losses both in terms of qualitatively as well as in
quantitatively.
Keywords: multiple PCR, nucleotide, primer, Co-PCR, PAGE, biosensors
Introduction
Several plant pathogens like fungi, bacteria, viruses, viroids, mollicutes,
etc. are responsible for disturbing crop yield as they cause severe plant
diseases and the rapid growth of the human population forces us to produce
additional food, but higher crop production also leads to the rapid spread of
disease. According to approximate plant pathogens such as fungi, viruses,
and bacteria cause a reduction in crop yield by 65%, 20% and 10%
respectively (Hansen and Lane, 1985; Khiyami et al., 2014), and the huge
losses of crops due to plant-pathogen infestation has been assessed to be
about 200 billion US$/annum (Khiyami et al., 2014). Identification of
injurious viruses, bacteria, and fungi in plant material, vectors/natural
reservoirs is important to ensure safe and sustainable agriculture (Lo´pez, et
al., 2003), and detection of plant pathogens showing symptoms can be
comparatively simple provided on has extensive experience with disease

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diagnosis and isolation of plant pathogens and accurate identification of
plant pathogens is one of the most important strategies for controlling plant
disease to initiate curative or preventive measures. Generally, viruses are
very small compared to other groups of plant pathogens like fungi and
bacteria which can be visualized through microscopes except plant viruses
are too much small to detect using light microscopes and they can be seen
only by a transmission electron microscope (TEM) and are made of a coat
protein and a type of nucleic acid (NA), either DNA and RNA based on the
nucleic acid core carrying hereditary information (Ellis et al., 2008). Plant
pathology deploys a broad range of techniques that do not give an adequate
solution to the entire disease losses in the case of viroids but innovative
molecular techniques enable fast and sensitive detection of viroids from a
combination of different techniques that gives the most reliable recognition
(Gucek et al., 2017).
New variants of polymerase chain reaction (PCR), like simple or
multiplex PCR in a single close tube, co-operative PCR (Co-PCR), and real-
time monitoring of amplicons or quantitative PCR, permit high sensitivity in
the detection of one or several pathogens in a particular test. The techniques
give ways to screen for a broad range of agents in a single test (Field and
Wills, 1998). In general, these methods are much faster, more accurate, more
specific, and can be performed and interpreted by personnel with no
particular taxonomic expertise.
Molecular techniques
Next Generation Sequencing (NGS)
It is also known as massively parallel or deep sequencing and is a
powerful technology that allows the generation of massive amounts of
sequence data and read lengths to vary from 35 to 800 nucleotides depending
upon the platform. The broad-spectrum and impartial nature of the
technology makes it an expensive tool for plant-based metagenomics,
allowing the immediate screening and detection of populations of grafted
transmissible agents to include viruses (either RNA or DNA), phytoplasma,
viroid in the sample. It can be used to concurrently detect the plant viruses
such as sweet potato feathery mottle virus (SPFMV) and sweet potato
chlorotic stunt virus (SPCSV). They targeted tiny RNAs 20 to 24 nucleotides
from total RNA extracts from co-infected plants.
Fluorescence in situ hybridization (FISH)
Fluorescence in situ hybridization is a technique useful for detection that
combines the simplicity of microscopy examination and the specificity of

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hybridization Recently, it is used in the detection of plant pathogenic
bacteria and is dependent on the hybridization of DNA probes towards
species-species regions of bacterial ribosomes. They are mainly suitable as
diagnostic targets since ribosomal RNA contains functional sequences that
are general to all species except also sequences that are very specific to
individual species, and FISH only needs to recognized particular
information. The sensitivity of the FISH technique is corresponding to that
of amplification technologies in addition to, in theory, it can detect particular
cells of bacteria, and the detection level is 103 cells per ml.
Polyprobes
Herranz et al. (2005) was developed this technique for the polyvalent
detection of numerous plant pathogens in a single assay. It is resulted from
the cloning, in complementary partial sequences typically between 200 to
400 nucleotide residues, of different plant viruses or viroids later than the
promoter region of an RNA polymerase. It has been effectively useful for the
detection of an array of combinations of plant viruses and viroids affecting
different crops e.g. citrus trees, pome and stone fruits, grapevine,
ornamentals, and vegetables.
Probe mix
This is the earliest molecular hybridization approach used to detect
diverse plant viruses at the same time involved the combination of several
probes, every one specific for a different target in the same test. It has been
successfully useful for the phytosanitary certification of tomatoes in Italy.
This technique frequently used in the detection of five viruses affecting
carnation (Sanchez-Navarro et al., 1999) and two viruses affecting geranium
plants.
Polymerase chain reaction (PCR)
The PCR have been used for detecting as the novel standard of a wide
strain of different plant viruses and this technique employs a pair of synthetic
oligonucleotides, every hybridizing to a single strand of a double-stranded
DNA (dsDNA) target, with the pair on both sides of a region that will
exponentially reproduce. The hybridized primer acts as a substrate for a
DNA polymerase, which produces a complementary strand.
• dsDNA separation at a high temperature above 90 oC.
• Primer annealing at 50-75 oC.
• Optimal extension at 72-78 oC.

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 Fig 1: Steps of Polymerase Chain Reaction
The augmented DNA fragments will be removed by agarose gel
electrophoresis and bands are visualized by staining with ethidium bromide
and irradiation with UV light and the specificity of PCR testing is dependent
on the basic coverage sets used that can detect an individual plant virus
species inside that genus. Generally, it has been used for the detection of
DNA viruses like genera Geminivirus, Caulimovirus and Nanovirus. The
sensitivity level of PCR is 1-10 pg/ml.

Fig 2: Illustration showing DNA bands separated on a gel

Co-operational polymerase chain reaction (Co-PCR)
A novel PCR concept of high sensitivity for the augmentation of plant
RNA virus, bacterial targets from plant materials has been recently described

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(Olmos et al., 2002) and the Co- PCR technique can be performed easily in a
simple reaction depending upon the simultaneous action of three or four
primers and the reaction procedure consists of the simultaneous reverse
transcription (RT) of two different fragments from the similar targets, one
internal to the other, the production of four amplicons with the combination
of the two different pairs of primers, one pair external to the other, and the
co-operational action of amplicons for the production of the largest
fragment. This technique has been successfully used both in the metal block
and capillary air thermal cyclers have working for the detection of RNA
plant viruses from different genera and bacterium but by using only three
primers (Caruso et al., 2003). The sensitivity observed is at least 100 times
higher than the RT-PCR and equivalent to nested RT-PCR.
Multiplex PCR
It allows the immediate and sensitive detection of numerous DNA or
RNA targets in a particular reaction and PCR detection protocols can be
designed to confirm the presence of the different pathogen in plant material
by looking for frequent specific sequences in two or more of them or detect
related viruses or bacteria on several hosts.

Fig 3: The image represents the multi-template multiplex PCR

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 Multiplex PCR is valuable in plant pathology since different bacteria or
RNA viruses frequently infect a particular host and thus sensitive detection
is essential for the production of pathogen-free plant materials.
Real-time quantitative PCR
Its uses two primers and a further dual-labeled fluorogenic probe to
permit the continuous monitoring of amplicon production during
thermocycling and require no post-PCR sample handling for target
quantification, real-time quantitative PCR to allow quick, sensitive detection
and monitoring of Helminthosporium solani on potato tubers.

Fig 4: Real-Time Polymerase Chain Reaction

Nested PCR
Nested PCR is used to attain a high level of specificity and sensitivity, it
is 1000 times greater than a particular PCR for fungal detection. In this
technique, two successive rounds occur in which a particular pair of primers
is used to increase a large region of DNA, then this amplified sequence of
DNA acts as a target for the next round by using two internal primers.
Generally, two species of Phytophthora; P. parasitica and P. palmivora
were detected by nested PCR.

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 Fig 5: Schematic representation of the two primer sets used in nested PCR
Loop-mediated isothermal amplification (LAMP)
It is performed at a constant high temperature for one hour using the
four primers. The loop formation is the first product and DNA has
continually amplified from the first products results in different size of DNA
structure and the LAMP product from the reaction can be detected through
electrophoresis, observed a spread of many bands in a lane of positive
LAMP reaction. It has been recently used for the simple monitoring of RNA
viruses including potato virus Y (PVY) and potato leafroll virus.
Nucleic acids sequence-based amplification (NASBA)
NASBA, a primer-dependent constant amplification and it has been used
for the direct amplification of RNA through polymerase chain reaction
(PCR) using reverse transcriptase, T7 RNA polymerase, and RNase H.
NASBA has been used to detect strawberry vein banding virus (SVBV) and
apple stem pitting virus (ASPV).
Biosensors
The work of biosensors is to sense the plant pathogen based on
biological detection with different receptors such as antibodies, phages,
DNA probes, etc. recognized a new biosensor for rapid diagnosis of soil-
borne pathogens, and this method was designed using two, unlike microbes,
each of them was immobilized on an electrode separately. Recently,
biosensors called affinity biosensors have been developed that use fragments
of nucleic acid as the basis for the recognition of plant pathogen. DNA-based
biosensors facilitate early detection of disease incidence before the

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emergence of a few observable signs or disease symptoms and it’s dependent
on the nucleic acid hybridization of DNA probe and is immobilized on
biosensors and analyte DNA sequence. Particular DNA sequences are widely
used for detecting fungi, bacteria, and genetically modified organisms
(GMO). Currently, a new type of biosensor based on bacteriophages is
emerging as a promising alternative for detecting pathogens due to their high
selectivity, thermostability, sensitivity and economics and it has been
successfully useful for controlling Dickeya solani (bacteria infecting potato
and tomato crops). It also used for detection and diagnosis of Pseudomonas
cannabina pv. alisalensis caused bacterial blight on radish.
Restriction fragment length polymorphism (RFLP)
It involves restriction enzyme incorporation of the pathogen DNA,
followed by separation of the fragments through electrophoresis in agarose
or polyacrylamide gels towards detect differences in the sizes of DNA
fragments. Polymorphisms in the restriction enzyme cleavage sites are used
to differentiate fungal species. While DNA restriction profile can be directly
observed with staining the gels, south blot analysis is usually essential and
DNA must be transferred to sufficient membranes and hybridized with a
suitable probe.
RFLP has been used for soil fungi and the study of the diversity of
mycorrhiza (Martinez-Garcia et al., 2011). It is also used for the
differentiation of plant pathogenic fungi (Hyakumachi et al., 2005).

Fig 6: Steps Involved in Restriction Fragment Length Polymorphism

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Amplified fragment length polymorphism (AFLP)
It analysis consists of the use of restriction enzymes to digest whole-
genome DNA, after that ligation of restriction half-site of particular adaptors
to everyone restriction fragments. Subsequently, selective amplification of
these restriction fragments is performed with PCR primer that contains in
their 3’ end the parallel adaptor sequence and discriminating bases. The
banding pattern of the amplified fragment is visualized on denaturing
polyacrylamide gels (PAG). The AFLP technology can amplify from 50 to
100 fragments at a particular time also to detect different polymorphisms in
diverse genomic regions simultaneously. It has been used to distinguish
fungal isolates at numerous taxonomic levels e.g. Aspergillus carbonas from
Aspergillus ochraceus (Schmidt et al., 2004) and to separate non-pathogenic
strains of Fusarium oxysporum from F. commune. AFLP markers have been
used to build up genetic linkage maps. (Van der Lee et al., 1997). AFLP
bands may also be used for SCAR markers development used in PCR-
dependent diagnostic tests.
Random amplified polymorphic DNA (RAPD)
Its analyses rely on PCR amplification of the pathogen genome by way
of small random sequences that are used as primers. These primers are most
likely able to find locality complementary sequences in the genome
producing particular banding patterns. The resulting PCR fragments are then
broken up by electrophoresis to get fingerprints that may differentiate fungal
species.

Fig 7: Randomly Amplified Polymorphic DNA

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 The specific DNA fragments identify in a profile may be cut out of the
gel and sequenced to attain a Sequence-characterized amplified region
(SCAR), into which specific primers can be intended for a more accurate
PCR recognition. SCAR primers have been used for example to particularly
identify Phytophthora cactorum (Causion et al., 2005) and Guignardia
citricarpa (Stringari et al., 2009).
Polyacrylamide gel electrophoresis (PAGE)
It has been a vital tool in the detection of unknown viroids because
information regarding their nucleotide sequences is not essential for the
detection of viroids. Various protocols connecting non-denaturing or
denaturing situation facilitate gel parting of viroids not only according to
their size but moreover according to their shape (linear or circular) and strain
(gentle/severe), which has enabled the progress of different protocols for
viroid detection. Sequential PAGE (S-PAGE) was earliest described by
Rivera-Bustamante et al., 1986 and has been the major tool for the
characterization of citrus and grapevine viroids (Duran-Vila et al., 1988).

Fig 8: Schematic diagram of polyacrylamide gel electrophoresis

It has been widely used for the conformation of viroid infection
(Malfitano et al., 2005), also for viroid identification, and has enabled the
separation and recognition of mild and severe strains. This technique is
based on temperature-related conformation changes of the rounded viroid
RNA and has been mostly used for the detection of CSVd (chrysanthemum)
and PSTVd (potato). Recently, the modification of the different PAGE
protocols has revealed that this technique is easy to perform, appropriate for
large-scale testing, versatile, and, in combination by way of silver staining,
still highly sensitive and specific (Hanold et al., 2003).

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Conclusion
Molecular techniques are effective tools for the detection of plant
pathogens and detection is the first step of plant disease management. These
techniques are also useful for the development of resistant varieties against
plant pathogen through the identification of specific genes or genotypes that
are present inside the pathogen. Molecular techniques are much faster,
highly sensitive, more accurate, more specific, and less time-consuming
compare then other laboratory techniques. They detected a pathogen without
producing any disease symptoms on the plant and identify a pathogen in its
latent period also.
References
1. Aslam S, Tahir A, Aslam MF, Alam MW, Shedayi AA, Sadia S. Recent
advances in molecular techniques for the identification of
phytopathogenic fungi-a mini review. Journal of Plant Interactions.
2017; 12(1):493-504.
2. Bernreiter A. Molecular diagnostics to identify fungal plant pathogens-a
review of current methods. ECUADOR ES CALIDAD-Revista
Científica Ecuatoriana, 2017, 4.
3. Borman AM, Linton CJ, Miles SJ, Johnson EM. Molecular
identification of pathogenic fungi. Journal of Antimicrobial
Chemotherapy. 2008; 61(1):i7-i12.
4. Gucek T, Trdan S, Jakse J, Javornik B, Matousek J, Radisek S.
Diagnostic techniques for viroids. Plant Pathology. 2017; 66(3):339-
358.
5. Jeong JJ, Ju HJ, Noh J. A review of detection methods for the plant
viruses. Research in Plant Disease. 2014; 20(3):173-181.
6. López MM, Bertolini E, Olmos A, Caruso P, Gorris MT, Llop P et al.
Innovative tools for detection of plant pathogenic viruses and bacteria.
International Microbiology. 2003; 6(4):233-243.
7. Makkouk KM, Kumari SG. Molecular diagnosis of plant viruses. Arab
Journal of Plant Protection. 2006; 24(2):135-138.
8. Schaad NW, Frederick RD. Real-time PCR and its application for rapid
plant disease diagnostics. Canadian journal of plant pathology. 2002;
24(3):250-258.
9. Tewari S, Sharma S. Molecular techniques for diagnosis of bacterial
plant pathogens. In Microbial diversity in the genomic era. Academic
Press, 2019, 481-497.

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10. Umesha S, Raghava S. Advanced molecular diagnostics for detection of
plant pathogenic bacteria. Indian Phytopathology, 2021, 1-6.

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 Chapter - 6
Role of Silicon in Plant Disease Management

Author
Jasmin Thomas
Ph.D. Student, Department of Plant Pathology, S.V
Agricultural College, Tirupati, Andhra Pradesh, India

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Page | 120
 Chapter - 6
Role of Silicon in Plant Disease Management
Jasmin Thomas

Abstract
Silicon is the second most abundant element (about 27.7% by mass) after
oxygen, accounting for almost 90% of the Earth's crust. Plants absorb silicon
from the soil solution in the form of monosilicic acid (0.1-0.6 mM), and it can
be regarded as a plant nutrient. The absorbed silicon accumulates in the old
tissues of the plants mainly in the walls of epidermal cells as polymerized
monosilicic acid or amorphous silica, which strengthens cell wall and
increases the structural rigidity of tissues. It plays an important role in
mitigation of adverse effects of abiotic and biotic stresses, promoting growth
and yield of crop.
The effect of silicon on plant disease resistance is thought to be caused by
either a build-up of absorbed silicon in epidermal tissue or the production of
metabolic or pathogenesis-mediated host defence mechanisms. Silicon can
control diseases such as blast to the same general degree as a fungicide and
reduce the amount and frequency of fungicides needed, reducing costs, and
providing positive environmental benefits. Silicon sources have residual
activity that persists over time, raising the possibility that applications are not
necessary on an annual basis. Cultivars that have lost resistance to a disease
such as blast and have good to excellent agronomic traits might again be useful
commercially, simply by using Si fertilization or amendments. Therefore, Si
sources and their management practices should be developed and practiced in
Integrated Disease Management programs.
Keywords: silicon, integrated disease management, biotic and abiotic stresses
Introduction
Silicate minerals make up more than 90% of the Earth's crust, making
silicon the second most abundant element in the crust (approximately 27.7%
by mass) after oxygen. (Epstein, 1991) [1]. Due to its high chemical affinity
for oxygen, it is found in a variety of forms in nature, including silica (SiO 2)
and silicates (SiO3), but only rarely in its elemental form. Plants absorb silicon

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from the soil solution in the form of monosilicic acid (0.1-0.6 mM), and it can
be regarded as a plant nutrient. The concentration of Silicon in plants on the
basis of dry weight varies between 0.1-10 percent. Silica concentration is
found to be higher in monocotyledons than dicotyledons. The absorption
capacity of different plant species may vary from 50 to 200 kg of Si /ha. The
absorbed silicon accumulates in the old tissues of the plants mainly in the walls
of epidermal cells as polymerized monosilicic acid or amorphous silica
(SiO2·nH2O), which strengthens cell wall and increases the structural rigidity
of tissues. Even though silicon (Si) is abundant in soil and plants, there is no
evidence that Si is needed for higher plants. Si, on the other hand, has been
shown to be beneficial to the growth of many plant species. It plays an
important role in mitigation of adverse effects of abiotic and biotic stresses,
promoting growth and yield of crop. Depending on the amount of biogenic
silica contained in their tissues, plants are classed as accumulators, excluders,
or intermediates [2]. Low accumulators (<0.1% Si) are Tomato, sunflower,
Gerbera etc, intermediary accumulators (1%) are Cucumber, Squash,
Chrysanthemum, marigold etc and high accumulators (more than 5% Si on
dry weight basis) are including Rice, sugarcane, Maize etc with respect to Si
absorption. Plant diseases are a significant threat to agricultural production
because they reduce crop yield and quality. Mineral nutrients have been shown
to increase or decrease plant resistance to pathogens and pests, and they are
essential factors in disease control.
In general, improved mineral nutrient management will reduce the
severity of the majority of plant diseases. This can be accomplished by altering
the availability of specific nutrients or increasing the efficiency with which
the plant absorbs and utilises them. The incidence of most diseases can be
minimised with proper nutrient management. There are several reports
available which shows the effectiveness of Si against different plant diseases
[3]
. The effect of Si on plant-microbe interactions and related physical,
biochemical, and molecular resistance mechanisms have been identified by
many researchers. Silicon accumulation in epidermal tissue, the creation of
complexes with organic compounds in cell walls, the activation of phenolic
compounds, phytoalexin/glucanase/peroxidase synthesis and controlling
pathogenicity or stress-related gene expression to limit pathogen invasion and
colonisation are all associated with the advantageous of Si on plant disease
resistance [4, 5, 6, 7].
Role of silicon in plants
Silicon promotes the growth of plants. The protection that Si provided to
plants allowed them to tolerate the negative impacts of abiotic and biotic

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stressors in many cases, resulting in increased growth. Silicon promotes cell
wall strength and rigidity, enhances growth, health, and productivity [8],
Increases the plant's ability to survive drought, frost, and salt stressors, reduces
lodging and increases the plant's resistance to insects, pests and disease
pathogens [9]. Many studies have shown that abiotic stresses cause greater
losses than biotic influences because the former cannot be regulated by
humans. Rice canopy photosynthesis is stimulated by Si because it increases
light interception by holding leaves erect [10]. To avoid mutual shading, this is
especially important in dense plant stands and when nitrogen fertilisers are
heavily applied.
Sources of silicon
Crop residues, especially of silicon-accumulating plants such as rice, are
used as silicon sources. However, the crop demand for application of Si
fertilizer generally exceeds that which can be supplied by crop residues.
Because of the low solubility of silicon, inorganic minerals like as quartz,
clays, micas and feldspars are poor silicon-fertilizer sources, despite their high
Si content. One of the most commonly used silicon fertilisers is calcium
silicate, which is a byproduct of steel and phosphorus manufacture. Potassium
silicate, though expensive, is highly soluble and can be used in hydroponic
culture. Other sources that have been used commercially are calcium silicate
hydrate, silica gel and thermo-phosphate. Different crops use Si minerals,
calcium silicate hydrate, silica gel, thermo-phosphate, diatomite, rice straw,
rice hull, rice hull ash, sugarcane bagasse and other Si-rich crop residues11.
Despite the abundance of soluble Si in most mineral soils around the world,
deficiency can occur as a result of Si depletion caused by continuous
cultivation of crops that require high amounts of Si, such as rice.
Why silicon in IDM
 Control several economically important diseases threatening one
crop, whereas some fungicides do not have as broad a spectrum of
activity.
 Silicon can control diseases such as blast to the same general degree
as a fungicide and reduce the amount and frequency of fungicides
needed.
 Reducing costs.
 Providing positive environmental benefits.
 Silicon sources have residual activity, so that applications are not
necessary on an annual basis.

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  Cultivars of lost resistance to a disease such as blast and have good
to excellent agronomic traits might again be useful commercially
simply by using Si fertilization or amendments.
 Cultivars that have high levels of genetic resistance to blast or other
diseases might still show yield increases due to Si effect on
agronomic traits.

Fig 1: Physiological role of Silicon in plants [12]

Source: Takahashi et al. 1990
Mechanisms involved in silicon-mediated disease resistance against
pathogens
Generally, mechanisms involved in silicon-mediated disease resistance
against pathogens can be classified into three types; physical, biochemical and
molecular mechanisms. Wang et al., (2017) has reviewed the effects of silicon
on plant disease and related resistance mechanisms [13].
Physical mechanisms
Silicon builds up in plant cell walls, forming a Silicon-cellulose network
that serves as a mechanical barrier against pathogens [14]. Silicon-enhanced
resistance is connected with the density of silicified long and short epidermal
cells, the thick layer of silica beneath the cuticle, the double cuticular layer,
the thickened Silicon cellulose membrane, the creation of papilla and

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complexes produced with organic compounds in epidermal cell walls that
mechanically reinforce plants.

Fig 1: (A) Leaf blast symptoms in rice after inoculated with Magnaporthe grisea for
10 days. Rice plants were continuously treated with (+Si) or without silicon (–Si). (B)
Silica layer was formed in the cell wall of Si-treated plants and enhanced plant
resistance to fungi infection by physical barriers [15]
Source: Sun et al., 2010

Crop Pathogen Physical Mechanism

Thick layer of silica is formed beneath
Pyricularia grisea
the cuticle of leaves and sheaths, form
Rice Rhizoctonia solani
complexes with organic compounds
Pyricularia grisea Silicified epidermal cell walls
Bitter gourd, Pythium Silicified epidermal cell walls in
Tomato aphanidermatum leaves
Rose Podosphaera pannosa Papillae formation
Grape Uncinula necator Si layer formation in the leaf cuticle
Cucumber, melon
Sphaerotheca fuliginea Polymerization of silicon on the leaf.
and pumpkin
Formation of a thicker epicuticular
Coffee Cercospora coffeicola
wax layer
Puccinia Thick layer of silica is formed beneath
Sugarcane melanocephala the cuticle of leaves
Leptosphaeria Sacchari

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Biochemical mechanism
Silicon-enhanced biochemical resistance is linked to (1) increasing the
activity of defense-related enzymes including polyphenol oxidase, glucanase,
peroxidase and phenylalanine ammonia-lyase (PAL), (2) inducing the
development of antimicrobial compounds like phenolic, flavonoids,
phytoalexins and pathogenesis-related (PR) proteins in plants and (3)
regulating systemic signals, such as salicylic acid (SA), jasmonic acid (JA)
and ethylene (ET) [16].
Crop Pathogen Biochemical Mechanism
Golovinomyces Higher levels of salicylic acid, jasmonic acid, and
Arabidopsis
cichoracearum ethylene
Cucumber Pythium spp. Chitinase, peroxidases and polyphenol oxidases
Mycosphaerella Increased activity of chitinase and β-1,3-glucanase
Pea
pinodes
Blumeria graminis f. Enhanced peroxidase activity in leaves
Wheat
sp. tritici
Accumulation of glucanase, peroxidase,
Rice Pyricularia oryzae polyphenol oxidase and phenylalanine ammonia-
lyase
Corynespora Chitinases, β-1-3-glucanases, PAL, peroxidases,
Soybean
cassiicola polyphenol oxidases
Ryegrass Magnaporthe oryzae Peroxidase and polyphenol oxidase
Colletotrichum Superoxide Dismutase, ascorbate peroxidase and
Bean
lindemuthianum glutathione reductase
Chitinase, superoxide dismutase, peroxidase and
Melon Podosphaera xanthii
β-1,3-glucanase
Increase in the concentration of lignin,
Rice Magnaporthe grisea antimicrobial glycosylated phenolics, diterpenoid
phytoalexins and lignin
Accumulation of phenolic substances impeded the
Pythium ultimum
penetration of into the vascular system
Cucumber
Increased flavonoid phytoalexin aglycone
Podosphaera xanthii
rhamnetin
Blumeria graminis f. Enhanced glycosylated phenolics and lignin
wheat
sp. tritici activities in epidermal cells

Molecular mechanisms
Silicon can function in the primary response and regulate the expression
of defence genes involved in cell wall structural modifications,
hypersensitivity responses, hormone synthesis, antimicrobial compound
synthesis and PR proteins [17].

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 Crop Molecular Mechanism
Expression of a protein rich in proline together with the
Cucumber
presence of silica at the site of pathogen penetration [18].
Differential regulation of 221 genes compared to untreated
Rice
control, including some transcription factors [19].
Potentiator of defense responses or as an activator of protein-
Wheat
mediated cell signaling [20].
Differential and unique expression of a large number of
Tomato, Rice,
genes involved in host plant defense mechanisms or
Arabidopsis and Wheat
metabolism [20].

Conclusion
Si plays significant roles in a plant’s life especially in alleviating both
abiotic and biotic stresses. It also promotes the growth and yield of the crop.
The mechanisms mediated by Si against pathogens are physical,biochemical
and molecular level. Si plays an important role in IDM. The reduction in
symptom expression is due to the effect of Si on components of host
resistance, including incubation period, latent period, lesion size, lesion
number, and inoculum production per infection site. The use of Si plus
reduced rates of fungicides are as effective as full rates of fungicides alone.
The number of fungicide applications and their rates can be reduced. The
residual activity of silicon was effective for disease control in the second year
crop and was comparable to a first year silicon application or a full rate of a
fungicide. It also provides positive environmental benefits. In addition,
cultivars that have high levels of genetic resistance to blast or other diseases
might still show yield increases due to Si effect on agronomic traits. Therefore,
Si sources and their management practices should be developed and practiced
in Integrated Disease Management programs.
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