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Is modulating virus SIR

Plant Science 234 (2015) 1–13
Contents lists available at ScienceDirect
Plant Science
journal homepage: www.elsevier.com/locate/plantsci
Is modulating virus virulence by induced systemic resistance realistic?
Franco Faoro a,b,∗ , Franco Gozzo c
Department of Agricultural and Environmental Sciences, University of Milan, Via Celoria 2, 20133 Milano, Italy
CNR, Institute for Sustainable Plant Protection, Strada delle Cacce 73, 10135 Turin, Italy
Department of Food, Environmental and Nutritional Sciences, Section of Chemistry and Biomolecular Sciences, University of Milano, Via Celoria 2,
20133 Milano, Italy
a r t i c l e
i n f o
Article history:
Received 8 October 2014
Received in revised form 17 January 2015
Accepted 20 January 2015
Available online 3 February 2015
Alternative oxidase
Hypersensitive response
Induced systemic resistance
Plant growth-promoting rhizobacteria
Systemic acquired resistance
a b s t r a c t
Induction of plant resistance, either achieved by chemicals (systemic acquired resistance, SAR) or by
rhizobacteria (induced systemic resistance, ISR) is a possible and/or complementary alternative to manage virus infections in crops. SAR mechanisms operating against viruses are diverse, depending on the
pathosystem, and may inhibit virus replication as well as cell-to-cell and long-distance movement. Inhibition is often mediated by salicylic acid with the involvement of alternative oxidase and reactive oxygen
species. However, salicylate may also stimulate a separate downstream pathway, leading to the induction
of an additional mechanism, based on RNA-dependent RNA polymerase 1-mediated RNA silencing. Thus,
SAR and RNA silencing would closely cooperate in the defence against virus infection. Despite tremendous
recent progress in the knowledge of SAR mechanisms, only a few compounds, including benzothiadiazole
and chitosan have been shown to reduce the severity of systemic virus disease in controlled environment
and, more modestly, in open field. Finally, ISR induction, has proved to be a promising strategy to control
virus disease, particularly by seed bacterization with a mixture of plant growth-promoting rhizobacteria.
However, the use of any of these treatments should be integrated with cultivation practices that reduce
vector pressure by the use of insecticides, or by Bt crops.
© 2015 Elsevier Ireland Ltd. All rights reserved.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Systemic acquired resistance against plant viruses: an uncertain fight against many targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Hypersensitive response-inducing viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Systemic viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Alternative respiration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Proteins involved in defense against viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Systemic acquired resistance and post transcriptional gene silencing: complementary or alternative defense strategies? . . . . . . . . . . . . . . . . 5
Induction of resistance to systemic viruses: a feasible practical approach? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
BTH (benzo[1,2,3]thiadiazole-7-carbothioate S-methyl ester, syn. acibenzolar-S-methyl) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Chitosans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Other potentially effective compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Plant-growth-promoting rhizobacteria (PGPR) and other beneficial microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Concluding remarks and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
∗ Corresponding author at: Department of Agricultural and Environmental Sciences, University of Milan, Via Celoria 2, 20133 Milano, Italy. Tel.: +39 0250316786;
fax: +39 0250316781.
E-mail address: franco.faoro@unimi.it (F. Faoro).
0168-9452/© 2015 Elsevier Ireland Ltd. All rights reserved.
F. Faoro, F. Gozzo / Plant Science 234 (2015) 1–13
1. Introduction
Virus diseases account for about half of crop epidemics [1].
Their control is mainly based on prevention by using genetically
resistant plants and by vector eradication, the latter implying high
costs and heavy environmental impact. In fact, no effective antiviral
compounds are available at present for field application [2]. Unfortunately, genetic resistance, either achieved by the conventional
introduction of Mendelian genes [3] or by genetic engineering
[4] can be overcome by viruses because of their genomic plasticity, and as it is very often based on gene-for-gene interaction
[5], recently reviewed [2,6]. The possibility of inducing resistance
in plants against viruses with chemicals or beneficial microorganisms deserves even more interest than that against bacteria and
fungi, because there are alternative workable strategies for these
Conventionally, there are two forms of induced systemic
resistance in plants. One is activated by numerous strains of plantgrowth-promoting rhizobacteria (induced systemic resistance, ISR)
and depends on hormones such as jasmonic acid and ethylene
[7]. The best known systemic acquired resistance (SAR) is induced
following a primary infection, particularly by pathogens inducing hypersensitive response [8], and, for practical purposes, can
be mimicked by the use of chemicals [9,10]. In Arabidopsis biologically activated SAR involves the expression of a number of
up-regulated genes, some of which are SA-independent while others are functionally associated with SA-depending defences and
pathogenic-related (PR) proteins. Most of all up-regulated genes
have been found to be up-regulated also by exogenous application
of chemicals, such as benzothiadiazole [11]. Nevertheless, also in
view of the relevance that diverse pathosystems may have on genes
activation, the systemic resistance induced by exogenous chemical
inducers cannot be necessarily authenticated as SAR on the basis of
the mere expression of a handful of genes. An updated insight into
SAR mechanisms, associated pathways, metabolites and epigenetic
modifications has been highlighted in recent reviews [12–15].
Both ISR and SAR are a condition of alerted defence that provides
long-lasting, broad spectrum resistance, which is effective against
different pathogens, including viruses [15]. The exploitation of ISR
against virus diseases has been less investigated [16–19]. There is
conflicting evidence whether ISR is really effective against these
pathogens [20].
In this review we pursue two main tasks:
(1) To expose evidence of the mechanisms responsible for preventing virus infections under controlled conditions. This task is
mainly covered by the biochemical induction of SAR and the
plant innate immune response involving RNA silencing, a predominant mode of basal plant defence against viruses [21].
(2) To ascertain the limits of defence against viral diseases in open
field, induced by application of the so far available chemical
inducers and biocontrol agents. In order to answer the question in the title, virus–host compatible interactions have been
selected as the most difficult to control.
2. Systemic acquired resistance against plant viruses: an
uncertain fight against many targets
2.1. Hypersensitive response-inducing viruses
The phenomenology of SAR was demonstrated for the first time
in 1961 by using the pathosystem tobacco mosaic virus (TMV)Nicotiana tabacum cv. Samsun NN [22]. The hypersensitive response
(HR), following TMV inoculation, triggered systemic resistance to
a subsequent challenge inoculation with the same virus, or other
Fig. 1. Different type reactions of tobacco to viruses. (A) Tobacco Samsun NN that
recognizes tobacco mosaic virus (TMV) triggering a hypersensitivity response (HR)
that localizes the virus into necrotic lesions. This prevents systemic infection. (B)
Compatible reaction of Tobacco White Burley that is not able to recognize TMV and
is infected systemically, leading to plant death.
unrelated necrotic viruses (see a detailed historical account in [23]).
It soon became evident that SAR was much more effective against
viruses producing localized infection, i.e. following HR response,
than against compatible viruses able to colonize the whole plant
(Fig. 1) [24]. The reason of this apparently diverging behavior
requires further insights. The diverse collection of PRs so far isolated do not appear to include antiviral agents, with the possible
exception of PR-10, a 18 kDa ribonuclease from Capsicum annum,
able to degrade TMV RNA [25]. Moreover, other events associated
with SAR induction, such as cell wall fortification and phytoalexin
synthesis, while effective against bacterial and fungal pathogens, do
not prevent virus replication or spread [24]. Thus, the increase of
endogenous salicylate would have to be the main defence response
to inhibit virus replication, cell to cell and long distance movement
of the viruses, which it does not always do [26–29]. It is not surprising that chemical-triggered SAR is only very effective against
HR-inducing viruses: in such a case the plant is, to a certain extent,
already “per se” resistant, being able to recognize a viral effector.
Thus, a chemical treatment only primes the plant to respond more
rapidly to the challenging inoculation, accelerating HR and, in turn,
reducing cell-to-cell spreading of the virus and the number of cells
involved in programmed cell death (PCD, Fig. 2) [27]. This, ultimately, results in the mitigation or lack of macroscopic symptoms.
So, challenging a plant with an HR-inducing virus still remains one
of the simplest methods to verify SAR establishment that can also
be quantified by counting the number and measuring the size of
necrotic local lesions produced on resistant and control leaf tissues.
The cell-to-cell virus spreading and loading into phloem can
also be delayed by callose deposition in plasmodesmata, either by
enhancing it synthesis or by inhibiting its degradation through ␤1,3-glucane synthase and ␤-1,3-glucanase, respectively [reviewed
in 30,31], although viral proteins can counteract the activity of
these enzymes by maintaining channels opened [32]. Callose
deposition is mediated by abscisic acid (ABA) [33], which blocks
salicylate-inducible defence responses [34], therefore ABA may be
detrimental in limiting virus infection, unless callose deposition
occurs in an early phase of the pathogenesis process, such as the
case of HR-inducing viruses [35,36].
However, from an agronomical point of view the infection of
HR-inducing viruses is often insignificant, as the related necroses
actually represent an efficient form of resistance due to the recognition of a viral gene product by plant innate immune system [37,38].
The plant immune responses are classically formulated according to two lines of defence, interacting with pathogens following
F. Faoro, F. Gozzo / Plant Science 234 (2015) 1–13
Fig. 2. Hypersensitive reaction (HR)-inducing viruses are a useful tool to assess
the activation of local and systemic acquired resistance. (A) A Phaseolus vulgaris
leaf sprayed with a resistance inducer (chitosan) and after 3 days inoculated with
tomato bushy stunt virus (TBSV) does not develop visible symptoms; (B) a similar
leaf sprayed with water before virus inoculation shows typical necrotic lesions due
to HR. P. vulgaris is therefore “per se” resistant to this virus responding with HR after
recognition of TBSV viral effector P19 (see text) and chitosan priming does nothing
but accelerate defense response. As a consequence, in chitosan primed tissues HR is
restricted to a very few cells, thus resulting in invisible symptoms.
the well-known zigzag model [37]. The first line is assumed to
occur at the cell membrane surface when suitable pattern recognition receptors perceive pathogen- (or microbe)-associated patterns
(PAMPs or MAMPs), e.g. bacterial flagellin or fungal chitin, and
activate the so-called P(M)AMP-triggered immunity (PTI). Evolved
pathogens circumvent this basal defence line by delivering into host
cells effector molecules that R-proteins in next co-evolved plants
can recognize as avirulence (Avr) factors, restoring and amplifying a specific form of resistance. The result of this recognition is
the so-called effector-triggered immunity (ETI), also known as host
resistance, which very often leads to HR. However, when dealing
with viral diseases, the use of these terms is questionable because
viruses are intracellular pathogens devoid of patterns equivalent
to PAMPs/MAMPs which are typical of other pathogens. Moreover,
the virus entry into plant cells may typically occur by membrane
damages, thus recognition would only be entrusted to cytoplasmic
R-proteins as an ETI process triggered by virus-encoded Avr factors, i.e. replicases, coat proteins, movement proteins and so on.
A recent line of thought believes that to fit viruses into the zigzag
model the basal resistance equivalent of bacterial and fungal PTI
might be represented by the RNA silencing mechanism that is triggered by dsRNA produced during virus infection. Thus, viral dsRNAs
would act as PAMPs inducing a basal defence response that viruses
can overcome by encoding RNA silencing suppressors, which, in
turn, may trigger ETI when recognized [39–41].
However, the PTI–ETI framework applied to viral pathogens
needs further experimental evidences, as there are many other
players in the game that cannot be fitted into the model, i.e. virus
encoded proteins which behave as Avr and trigger HR but are not
silencing suppressors, or host co-factors that modify the specificity
of Avr recognition by R-proteins. An exhaustive and critical account
of virus recognition by plant immune system can be found in some
recent reviews [23,41,42].
2.2. Systemic viruses
Virulent virus diseases are produced only by compatible
viruses that are able to spread systemically (Fig. 1) and SAR
does not seem to be so effective in controlling them [43]. Some
compatible viruses can also cause systemic necroses, sometimes
leading to plant death [23]. Intriguingly, the development of these
systemic necroses apparently lacks fundamental differences in
genetics, biochemistry and physiology from the HR-associated
ones, that might justify the dramatic jump from the localized
lesion to the lethal systemic event. Indeed, some genes involved
in incompatible interactions are active, even if with different roles,
in systemic necroses investigated in Nicotiana benthamiana [44,45].
In this view, systemic necroses could be regarded as a last, possibly
useless, attempt to block a virus that escaped HR recognition [23].
Nevertheless, an impressive number of reactions and interactions
is emerging from the study of systemic viral infections, as will be
described below.
Up to the middle of 1990s only a few papers had taken notice of
SAR induction against systemic virus infections (see review [24])
in spite of the number of works published on this topic. Moreover,
the above mentioned studies did not provide convincing evidence
of a success, leading the authors of that review to the conclusion
that SAR does not operate against systemic virus infection [24].
Since then, the scenario has been changing a great deal and in the
last decade a number of studies demonstrated that SAR can also
be induced against systemic virus diseases, including for practical
uses [46].
Above all, the mode(s) of how salicylate is involved in induced
resistance to systemic viruses turned out to be intricate [28,29,47].
Salicylate inhibits virus replication at the site of inoculation in some
virus–host combinations, such as potato virus X (PVX)-tobacco
plants and Arabidopsis-turnip vein clearing virus (TVCV) [48]. In
other combinations, as TMV-tobacco Xanti nn (lacking N resistance
gene), it impairs both replication and cell-to-cell movement [49,50]
while in other cases, as cucumber mosaic virus (CMV)-tobacco or
CMV-Arabidopsis, salicylate does not inhibit the replication nor
the cell-to-cell movement but it interferes with systemic movement [51]. In these pathosystems salicylate interacts with the
virus-encoded multifunction protein 2b, which is a RNA silencing suppressor [52]. However, salicylate-induced resistance to CMV
appears due to the inhibition of virus replication in squash (Cucurbita pepo) [51].
These findings suggest that salicylate triggers resistance to at
least one of the three phases of the infection process (replication, cell-to-cell movement and systemic movement through the
veins), depending on the virus–host combination. This strategy,
coupled with RNA silencing mechanism, would ensure defence
against different viruses [28]. Again, systemic responses to viral
challenges, in common with infections by other pathogens, also
involve SAR signals mediated by several metabolites, including
glycerol-3-phosphate, azelaic acid, pipecolic acid, dehydrobietinal,
methyl salicylate and glycerolipids [53,54].
Progress in plant protection against pathogens, including
viruses, has been the result of thorough insights into SAR mechanisms, as well as the discovery of some chemicals that induce
SAR phenotype without triggering cell death [9,15,48]. These
include benzothiadiazole (BTH, syn. acibenzolar-S-methyl), the
well-known functional analogue of salicylate that effectively
induced resistance against a systemic TMV infection in tobacco
Xanthi “nn” reducing the viral RNA by >95% in both inoculated and
uninoculated upper leaves [55] without inducing HR. Evidence of
defence activation by BTH is corroborated by its induction of PR-1
expression, the main biochemical marker of the SAR transduction
pathway. This is activated at the site of, or downstream of salicylate accumulation, circumstantially inferred by the occurrence of
this effect even when salicylate cannot accumulate in the tissues
While the biologically induced SAR, i.e. following HR-triggering
pathogen infection, is believed to involve systemic signals to spread
defence to all systemic tissues, an analogous signal, even if present,
F. Faoro, F. Gozzo / Plant Science 234 (2015) 1–13
is not necessarily invocable for the systemic resistance induced by
BTH, which, unlike salicylate, is highly mobile. Therefore, BTH can
diffuse in all leaves independently on the localized or widespread
application and its action may be also considered as a localized
defence triggered in all leaves.
A peculiar process activated by chemical inducers of SAR occurs
during the so-called “priming”, a phase lasting weeks or months,
during which the plant becomes alerted to promptly exploit its
natural responses at the moment of pathogen challenge. The mechanism of this long lasting process remains to be clearly elucidated.
The principles of epigenetics have been recently applied as an
interesting approach to understand the mode of action of priming.
The BTH activation of the transcription gene WRKY 29, involved
in gene priming, was associated with histone chemical changes
[57]. The expression of this gene was however activated only when
the plants were challenged by an additional stress, such as infection by a pathogen [57]. These observations, together with other
evidence, support a hypothesis that long-lasting memory is maintained as a stable SAR signal. The approach was independently
elaborated by various research groups and led to the discovery of the trans-generational SAR, inherited by small interfering
RNA (siRNA)-directed DNA hypomethylation [14]. This mechanistic
model of SAR long-lasting persistence, even if through specifically
alternative processing, appears to be in some way operative in
infections by different pathogens, including viral, bacterial and fungal diseases (see [23]). It provides a rationale for understanding the
originally reported persistence for at least 3 weeks of the TMVinduced SAR, observed a half century ago.
To expose significant processes involved in viral infections,
other notions gained from the naturally induced SAR need to be
2.3. Alternative respiration
An interesting finding was the discovery that salicylate rises
alternative oxidase (AOX) [28], a mitochondrial enzyme that provides an alternative respiration by shortcutting the cytochrome
electron transport pathway that reduces molecular oxygen. This
occurs particularly during biotic and abiotic stresses [58]. AOX,
which is resistant to cyanide, regulates and limits the generation of reactive oxygen species (ROS). Treatment of tobacco with
salicylhydroxamic acid (SHAM), a selective inhibitor of AOX, abolishes salicylate-induced resistance to TMV, as well the salicylate
impairment of PVX replication and CMV movement, while not
inhibiting PR protein synthesis [49,59]. At the same time, salicylateinduced resistance to bacterial and fungal pathogens, i.e. Botrytis
cinerea and Erwinia carotovora, is not affected by SHAM. Some
researchers proposed that the SAR pathway branches downstream
of salicylate into two directions, one SHAM-sensitive, operating
against viruses, and the other SHAM-insensitive inducing PR proteins and resistance to bacteria and fungi [49]. A possible link
between AOX induction and induced resistance to viruses could
be the ROS modulation in mitochondria. Here an established role
has been formulated for redox signaling and ROS in plant defence
[60]. Moreover, the level of ROS in mitochondria may have a
part in the coordination of defence and stress-induced signaling.
Support for this theory relies on the evidence that resistance to
viruses in tobacco and Arabidopsis can be induced by using sublethal concentrations of antimycin A and cyanide [48,60] These
chemicals inhibit electron transport through the cytochromes raising the ROS level and, in turn, involving AOX. SHAM treatment
also enhanced a compatible TMV-tomato interaction resulting in
virus replication, which was reduced by cyanide [60,61]. In this
pathosystem, the mitochondrial AOX pathway was up-regulated in
inoculated leaves leading to less ROS production in the upper leaves
and to enhanced basal plant defences [60]. Moreover, promoter
Fig. 3. Scheme of how salicylate counteracts viruses in two manners. Salicylic acid
activates two different pathways that counteract virus infection: by stimulating
the transcription of RNA-dependent RNA polymerase 1 (RDR1) that mediates the
induction of RNA silencing and by inhibiting the respiratory electron transport chain
(dotted line) in mitochondria. This inhibition is assumed to involve components of
ROS, possibly produced by constriction of electron flow or by salicylate-interaction
with peroxidases. These components are detected by sensor proteins and the related
signal is transduced to the nucleus where it may induce both defense and alternative
oxidase (AOX) genes. AOX negatively regulates the amplitude and duration of ROS
generation. Inhibitors of the respiratory electron transport, such as antimycin A (AA),
cyanide (CN− ) and a strobirulin-derived fungicide (SB) induce ROS accumulation in
mitochondria as well, activating the expression of defense genes. Nitric oxide (NO),
a molecule generally associated with ROS, appears to be another mediator of AOX
accumulation, so contributing to limit viral infection.
Modified from Singh et al. (2004) [28].
sequence analysis of tomato plants showed noticeable similarity
between AOX1 promoter and promoters of PR-1 and GRP8 (glycinerich protein) genes, main participants in resistance responses to
pathogens [60]. Application of cyanide and a NO-donor (diethylamine nonoate) to tomato plants induced accumulation of AOX and
reduced TMV viral RNA. Conversely, application of SHAM or a NO
scavenger (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline1-oxyl-3-oxide) antagonized resistance to TMV [61]. The results
fit a model in which NO generation induced by TMV acts as a positive mediator of the AOX induction and this then modulates the
redox system of mitochondria, reducing ROS production and limiting systemic infection [61]. Interestingly, a strobirulin fungicide
that inhibits mitochondrial respiration also induces resistance to
virus infection without activating PR protein synthesis [62]. Other
correlative indications that AOX is involved in defence mechanisms
are: (i) its overexpression in HR-inducing interactions [59]; (ii) the
possibility of activating either salicylate- or antimycin A-induced
resistance against viruses, but not against bacteria and fungi, in the
Arabidopsis nonexpressor of PR-1 gene (npr1) mutants impaired in
PR-1 synthesis [48].
The infection of transgenic tobacco plants in which AOX expression was increased or decreased, showed that salicylate, but not
antimycin A, can trigger an additional AOX-independent antiviral
mechanism based on the induction of a RNA-dependent RNApolymerase 1, leading to an increased viral RNA turnover [63].
A model summarizing the interactions described above is shown
in Fig. 3.
2.4. Proteins involved in defense against viruses
The most efficient defense response relies on R genes that
encode suitable proteins capable of recognizing viral elicitors [38].
Many of these R-proteins conserve a domain characterized by
F. Faoro, F. Gozzo / Plant Science 234 (2015) 1–13
leucine-rich repeat motif sequences. Other domains cooperate
with leucine-rich repeat sequence proteins conferring successful
resistance activation [64]. Lectins, that generally bind to glucan molecular matrices, are also involved in viral resistance. A
peculiar Arabidopsis Jacalin-type lectin, RTM1 (RESTRICTED TEV
MOVEMENT 1) mediates resistance to tobacco etch virus (TEV)
[23]. A similar protein, encoded by the lectin gene JAX1, promotes resistance to poty viruses. This type of lectin-mediated
resistance apparently does not depend on HR or SAR signaling
[65]. This is quite unlike the resistance mediated by the nucleotide
binding site-LRR (NB-LRR) proteins, which are encoded by R
genes. Two other lectin-like proteins were isolated from Cyamopsis tetragonoloba after elicitation by CIP-29, a systemic resistance
inducing protein from Clerodendrum inerme [66]. These basic glycoproteins, CT-VIA-32 and CT-VIA-62, impaired virus infection in
several virus–host combinations when inoculated contemporaneously with the pathogen [66]. This behavior is reminiscent of
ribosome inactivating proteins (RIPs) that both directly inactivate
the virus and impair its synthesis by hydrolyzing the N-glycosidic
bond of a specific adenosine of the major rRNA [67]. In fact, Dioicin
2, a typical RIP from Phytolacca dioica, had a strong antiviral activity,
expressed only when both RIP and virus were inoculated together
in the same leaf (either on the same surface, or separately in the
adaxial and abaxial surfaces) but did not induce SAR [68].
The RTM1 and RTM2 proteins, expressed mostly in the phloem
of A. thaliana Columbia (Col-0) plants, partially blocked the infection by three potyviruses, TEV, plum pox virus (PPV) and lettuce
mosaic virus (LMV). Some isolates of both PPV and LMV overcame
the RTM mechanism. The viral determinants interacting with this
type of resistance have been investigated using recombinant DNA
technology. The resistance determinants in both isolates were in
the N-terminal region of the coat protein, one of the three potyviral proteins involved in the long-distance movement [69]. These
changes in amino acid sequences suggest that interactions of RTM
proteins with viral coat proteins may be responsible of changes in
the resistance mechanism(s).
Ubiquitin proteins have acquired increasing importance as regulators of post-translation changes, giving out to the process called
ubiquitination that regulates degradation of cellular proteins by
the ubiquitin proteasome system, controlling a protein’s half-life
and expression levels. Critical steps of this process are mediated
by special enzymes, E1 , E2 and E3 , the last of which possess ligase activity. The plant ubiquitin proteasome system is frequently
involved in interactions between plants and pathogens, including
viruses, leading to plant resistance signaling. One of the first manifestations in tobacco is induction of ubiquitin-activating enzymes
NtE1 A and NtE1 B which are induced when the plants are inoculated with TMV or tomato mosaic virus (ToMV) [70]. E3 ubiquitin
ligases, the most abundant enzymes of the ubiquitination system,
are implicated in a variety of plant growth processes as well as in
defense against viruses through the proteasome machinery. However, ubiquitin proteasome processes may also promote viruses
virulence [23]. These roles of the ubiquitin proteasome machinery are multifaceted consequences of opposite strategies that may
be used by plants and viruses ultimately resulting in resistance or
susceptibility to infection [71].
2.5. Systemic acquired resistance and post transcriptional gene
silencing: complementary or alternative defense strategies?
The post transcriptional gene silencing, also known as RNA
silencing or RNA interference (RNAi), is a mechanism that both
plants and animals use to degrade foreign, over-expressed or aberrant RNA molecules in a sequence-specific manner. This involves
the processing of viral double-stranded RNA, degraded by Dicerlike (DCL) enzymes, i.e. DCL2 and DCL4, into siRNAs, 21–24
nucleotide (nt) RNA duplexes. These siRNAs are subsequently
incorporated into protein complexes that target and degrade viral
RNAs via the endonucleolytic activity of Argonaute proteins (AGO),
mostly AGO1 and AGO2 [41,72]. RNAi can be a powerful mechanism
in preventing virus infection but most viruses are able to encode
RNA silencing suppressors (RSSs) that overcome it [73]. The various
mechanisms by which RSSs operate have been recently reviewed
In the previous Section 2.1, virus silencing triggered by viral
dsRNA has been regarded as a basal defense response, analogous
to PAMP-triggered immunity (PTI) of bacteria and fungi, in spite
the fact that it is a RNA-based, rather than a protein-based, resistance mechanism as the typical PTI. Indeed, there is a functional
connection between salicylate-induced resistance to viruses and
RNA silencing triggered by dsRNA, being the latter also synthesized
by RNA-dependent RNA polymerases (RDRs), which, in turn, are
salicylate inducible [75] (see Section 2.3). However, in transgenic
tobacco plants where RDR activity had been compromised, these
enzymes could be dispensed with salicylate-induced resistance
as the related transgenic plants still showed enhanced resistance
to TMV after treatment with salicylate [75]. Nevertheless, RDR1,
though not necessarily required for salicylate-induced resistance,
has a major role in limiting virus accumulation in the infected
tissues. In fact, suppressing its inducible activity renders tobacco
plants more susceptible to TMV and PVX [75]. Therefore, RDR1 must
be considered a component of plant innate immunity [76] and the
fact that N. benthamiana is susceptible to many viruses is likely the
consequence of a natural mutation in its RDR1 gene that encodes
an inactive enzyme [77]. Other RDRs, such as RDR2 and RDR6 of
tomato are involved in antiviral silencing. RDR1 and RDR6 are transcriptionally activated following ToMV infection, while RDR1 and
RDR2 are activated by salicylate which, in turn, results in enhanced
resistance to ToMV [78]. Similar results have also been achieved by
treatments with gentisic acid, a metabolic derivative of salicylate,
though with slight different activation of polymerase types. The
same group had similar results with Gynura auriantiaca, infected
with citrus exocortis viroid (CEVd), with activation of different RDR
combinations. Thus, salicylate induced silencing has a major role
also in defense mechanism against viroids [78].
As above-reported, most viruses escape silencing through RSSs,
and the studies on these viral encoded proteins showed that
there is an overlapping of silencing and SAR signaling. A typical
example is PPV-encoded proteinase HC-Pro, a well-known multifunctional protein that, besides RSS activity [79], is involved in
aphid transmission of the virus, virus movement, induction of disease symptoms and interference with the activity of endogenous
regulatory microRNA (miRNA) [79–82]. PPV systemically infects
several tobacco species, such as N. benthamiana, Nicotiana clevelandii and Nicotiana occidentalis, but not N. tabacum, where it
remains localized in the infected leaves. PPV produces necrotic
lesions resembling HR on N. tabacum and induces salicylatedependent defense products, such as PR proteins, AOX and RDR
activity, but did not raise SAR in uninoculated leaves [82]. In N.
tabacum transgenic plants expressing the NahG gene, that impairs
the accumulation of SA, or the antisilencing sequence of P1/HCPro from TEV (a potyvirus that spreads systemically in tobacco),
PPV produces systemic infection. When both NahG and P1/HC-Pro
were co-expressed in the same plant, the PPV systemic movement
was enhanced, suggesting that there is an overlap between the signaling pathways that control SAR and the induction of silencing
[83]. PPV-derived siRNAs, a hallmark of RNA silencing, accumulate to lower levels in the NahG tobacco mutant plants than in the
wild-type tobacco. This correlates well with the accumulation pattern of the virus and the expression of the salicylate-induced RDR1
gene [83]. These data suggest that salicylate enhances viral RNA
turnover, acting as a positive regulator of RNA-silencing defense,
F. Faoro, F. Gozzo / Plant Science 234 (2015) 1–13
while the silencing suppressor HC-Pro alters regulation of the
salicylate-mediated defense response. The interference of P1/HCPro with the salicylate pathway has been demonstrated also in
pea lines infected with clover yellow vein virus (ClYVV). In this
case, mutations in P1/HC-Pro, while impairing the suppression of
RNA-silencing activity, reduced both virus virulence and salicylatemediated PR expression. Intriguingly, virulence could be partially
restored by BTH or exogenous salicylate application, suggesting
that high activation of the salicylate signaling pathway is required
for ClYVV infection [84]. Thus, it appears that salicylate signaling pathway has opposing functions in compatible interactions,
depending on the virus–host combination.
Many other RSSs are multifunctional proteins able to interfere with SAR pathway, sometimes in diverse modes, depending
on the virus–host pathosystem. Some of the most studied RSSs
are reported in Table 1 [39,52,79–83,85–95], together with the
counter–counter defenses that plants activate in the attempt of
controlling the infection, once that silencing mechanism has been
impaired. Some RSSs are also able to suppress salicylate-mediated
resistance, as the case of CMV 2b and this fact may explain the
wide host range (more than 1000 plant species, in 365 genera
from 85 families) and worldwide distribution of CMV, possibly the
most successful plant pathogenic virus [86]. The 2b protein is also
able to prime the induction of SA biosynthesis and, conversely,
to antagonize the jasmonate pathway, thus favoring aphid feeding in infected Arabidopsis plants, and, ultimately, virus spreading.
[96,97]. Inhibition of jasmonate-induced gene expression by 2b and
other suppressors of virus silencing does not always correlate with
enhanced aphid performance and other unknown factors must be
involved [98]. The impairment of both the main defense pathways,
besides explaining the wide CMV host range, again underlines the
notion that the evolutionary success of viruses mainly relies on
multifunctional encoded proteins [1].
Plants can even use a sort of HR-like rapid response as a second line of defense, to be activated only if the initial antiviral
response does not restrict virus levels below a threshold where
RNA translation remains below a sufficient level of viral protein for
Avr recognition. Thus, a true HR could not be displayed, despite
the fact that host-virus interaction would be competent to do it
[99]. This is the case of some resistance genes, e.g. Rx of potato,
that has a nucleotide binding site, a leucine-rich repeat (NBSLRR) class of plant R genes inducing HR. Potato Rx, expressed as
a transgene either in potato or in Nicotiana spp. confers extreme
resistance against PVX and this resistance is not associated with
HR, showing that the antiviral responses can be uncoupled from
Avr recognition [99]. Furthermore, when Rx-genotype protoplasts
are infected with PVX together with either TMV or CMV, viral RNAs
from both PVX and the second virus fail to accumulate [100], suggesting that, once initiated, antiviral responses indiscriminately
target viruses. Another example of extreme resistance is triggered
by P19, the RSS of TBSV in N. tabacum specific cultivars as a
plant counter-counter defense. This response involves the salicylate and ethylene pathways, as well as PR protein synthesis, without
causing HR [39].
As reported in Table 1, RSSs may induce HR in particular
host species where they are recognized as Avr by R-proteins.
This counter-counter plant defense can be regarded as a classical effector-triggered immunity (ETI) and in some cases requires,
as for bacteria, the mediation of DRB4, which is a dsRNA-binding
protein associated with a dicer-like protein 4 (DCL4) to produce virus-specific siRNA [95]. Another co-factor able to mediate
R-protein recognition of RSSs is catalase 3 whose affinity with
CMV 2b is responsible for the degree of necrosis in different
CMV-Arabidopsis ecotypes combinations [87,101]. The requirement of host co-factors, besides the presence of specific R-proteins,
explain the great variability among the host species to RSS-induced
Fig. 4. Chemicals tested or proposed as inducers of resistance against viral diseases.
BTH, benzothiadiazole; BABA, ␤-aminobutyric acid.
ETI response. Recently, it has also been demonstrated that plant
miRNAs are able to regulate R-gene expression through RNA silencing [reviewed in 102], possibly to avoid their overexpression in
absence of the pathogen. This mechanism is, in fact, inhibited upon
virus infection. Deep-sequencing studies (i.e. RNASeq) have led to
the discovery of miRNAs that can directly target host R-gene transcripts, such as miR1885 induced in Brassica rapa by turnip mosaic
virus (TuMV) that targets mRNA of R-genes encoding TIR-NB-LRR
receptors [103]. Other miRNAs target directly silencing components, such as DCLs, DRB4, RDR6 and Argonaute proteins [104].
This highlights the complexity of defense response against viruses,
peculiar pathogens which are able to redirect cellular synthetic
machinery in their favor and thus deserving many counteracting
strategies by the plant immune system.
3. Induction of resistance to systemic viruses: a feasible
practical approach?
For the reasons specified in the first part of this review, the
induction of resistance to HR-inducing viruses is a very useful
model for screening compounds able to activate SAR. Although
some of these viruses can promote secondary infections resulting
in severe foliage damages and yield losses, the most economically
important virus diseases are produced by systemic viruses [24].
Here we survey those few compounds that are effective in preventing or limiting this type of infection (Fig. 4). Furthermore, as
a virus is dependent on host and vector for its survival, the effects
of induced resistance on vector transmission are also discussed,
together with another important parameter that is the induction phase, regarded as the minimum time during which a host
plant needs to reach an adequate level of resistance. Finally, some
practical uses of plant-growth-promoting rhizobacteria (PGPR) in
inducing ISR against viruses are discussed.
F. Faoro, F. Gozzo / Plant Science 234 (2015) 1–13
Table 1
Virus RNA silencing suppressors and plant counter–counter defense.
Aphid transmission,
virus movement,
perturbation miRNA
metabolism, symptom
Silencing suppressor
Host plant
Infection type
Nicotiana benthamiana
N. clevelandii
N. occidentalis
N. tabacum
Cucumis sativum
N. glutinosa
N. tabacum
N. tabacum seedlings
Systemic only after SA
Arabidopis Col-0
Solanum lycopersicon
N. tabacum
N. sylvestris
N. bonariensis
N. tabacum specific cultivars
Extreme Resistance
defence factors
Other functions
HR-like, PRs, AOX,
2b dispensed for
HR(requires as
co-factor catalase
3), SA, H2 O2
P38 (CP)
T. Samsun NN
T. Xanti nc
Arabidopsis Di-17
HR (induced by P50
HR (CP recognition
requires DRB4 host
miRNA metabolism
and symptom
AOX, alternative oxidase; CP, coat protein; DRB4, dsRNA binding protein; ET, ethylene; JA, jasmonate; HR, hypersensitive reaction; PRs, pathogenesis related proteins; RDR,
RNA-dependent RNA-polymerase; SA, salicylate.
3.1. BTH (benzo[1,2,3]thiadiazole-7-carbothioate S-methyl ester,
syn. acibenzolar-S-methyl)
BTH (trademarks: BionTM in Europe, ActigardTM in USA), is
a functional analogue of salicylate. It was the first commercial
product to induce an artificial type of SAR against systemic virus
infection, e.g. TMV in tobacco Xanthi “nn” [55]. BTH inactivates
catalases (CAT) and ascorbate peroxidases (APX), the two major
H2 O2 scavenger enzymes, increasing the H2 O2 pool in treated tissues [10]. This may occur without inducing plant cell death when
BTH is applied in a suitable range of concentrations [105], that is
usually between 1 and 100 mg l−1 , depending on the host plant.
The much stronger activation of salicylate signaling pathway by
BTH (after an induction phase of 3–7 days), with respect to exogenous application of salicylate, and the much lower phytotoxicity
compared to the latter [55] are the good reasons for its successful utilization in the field against different pathogens, including
One of the most striking instances is the control of tomato spotted wilt virus (TSWV) in N. tabacum [106,107]. In this pathosystem
the most effective treatment was performed before transplantating [108]. After transplanting, a combination of BTH and the
insecticide imidacloprid proved to be more suitable to contain the
disease [107–109]. Such a combination is required because TSWV
is efficiently transmitted by eight species of two thrips genera,
Frankliniella and Thrips [110]. Moreover, TSWV replicates in thrips
vectors, thus the insect not only spreads the virus, but serves as a
virus host, in part explaining the wide number of plants that can
be infected (over 1000 plant species in 80 botanical families).
Traditionally, farmers try to control TSWV by applying broadspectrum insecticides on a calendar basis (see [111]). This approach
is costly, and has a high environmental impact as few insecticides
are efficacious against thrips, and some of these are carbamates or
organophosphate insecticides. These may not be available in the
near future because of restrictive rules on pesticides. The most
effective integration of different control strategies against TSWV,
CMV and potato virus Y (PVY) [112] was the use of UV-reflective
plastic mulch in combination with BTH and insecticide treatment
[113–116]. Similar management tactics controlled Bemisia tabaci
vectored tomato yellow leaf curl sardinia virus (TYLCSV) in protected tomato crops [114].
BTH, applied to watermelon and melon plants, induced systemic
resistance against zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV) and CMV [117] as well as cucurbit
chlorotic yellows virus (CCYV) vectored by B. tabaci [118]. The
analysis of plant total RNA by quantitative reverse-transcription
polymerase chain reaction (real-time RT-PCR) showed that accumulation of viral RNA was almost undetectable in lower and upper
leaves and greatly reduced in the intermediate ones. The reduction of viral RNA by BTH treatment strictly correlated with PR1
gene expression that was higher only 1 week post-inoculation. This
confirmed systemic resistance activation but also suggested that a
complete control of the disease required weekly treatments. Melon
plants sprayed once with 25 mg l−1 BTH were slightly stuntined
within 2–3 weeks, which may present an unacceptable fitness cost.
BTH-successfully elicited defense against bacterial and viral
pathogens has been evaluated in peppers under field conditions
[119]. The level of protection paralleled a clear effect of priming,
displayed by an increase in the expression of Capsicum annuum
PR4 (CaPR4) gene. The accumulation of CMV, detected by qRT-PCR
analysis, was significantly lower in BTH treated plants with respect
to control at 10 days after treatment, but by 40 days the difference was not so significant. In previous greenhouse experiments,
the use of CaPR4 as a priming marker in pepper had been found
F. Faoro, F. Gozzo / Plant Science 234 (2015) 1–13
Fig. 5. Structure of acetylated monomers of chitin and deacetylated chitosan units and most diffusable commercial available chitosans, broadly classified on the basis of
their molecular weight (m.w.) at the deacetylation degree of 80–85% and the corresponding viscosity in mPa·sec.
to be reliable, being consistently correlated with the SAR induced
by BTH. However, in the currently reported study, the reliability of
CaPR4 was limited to the results observed at 10 dpt. The expression of this gene did not correlate with disease defense at longer
times. The authors suggested that the exposure to environmental
and biological factors may alter the reliability of some genes, such as
CaPR4, and requires the need to identify other more sensitive markers. These comments are in agreement with the general feeling by
various experts that induction of SAR under open field appears to
be conditioned by the plant necessity to modulate defense genes
Fig. 6. Possible mechanism of local and systemic acquired resistance (LAR and SAR) induction by chitosan. Chitosan (stained in blue by toluidine) sprayed on a bean leaf
enters through stomata and causes a Ca2+ influx into mesophyll cells leading to callose deposition (bright fluorescence by aniline blue) and oxidative burst in the cells around
the sub-stomatal cavity (brown staining by 3-3 diaminobenzidine). Some of these cells encounter programmed cell death (PCD, stained in blue by Trypan); thus a network
of small groups of dead cells (micro-HR, encircled) is formed in the leaf mesophyll generating signals for LAR and SAR establishment (all bars = 30 ␮m).
F. Faoro, F. Gozzo / Plant Science 234 (2015) 1–13
Fig. 7. Different mechanisms of local and systemic acquired resistance induced by
BTH and chitosan. Both compounds induce resistance via an oxidative burst due to
H2 O2 deposition. However, in BTH-treated tissues, H2 O2 is uniformly localized in
epidermal cells at a sublethal level, without triggering a cell death program (PCD),
which is instead generated in chitosan treated leaves, due to its accumulation in the
sub-stomatal cavity. As a consequence PCD is triggered in the neighboring mesophyll cells with the formation of micro-lesions which mimic a HR-inducing virus
infection, in turn generating signals for local (LAR) and systemic resistance (SAR)
with the aim of minimizing multiple environmental adverse factors
Three consecutive treatments of pepper plants with BTH could
reduce significantly the infection by the geminivirus pepper golden
mosaic virus (PepGMV) throughout the season. [121].
3.2. Chitosans
Chitosans are natural, non-toxic and inexpensive products
obtained by deacetylation of chitin from the exoskeleton of crustacean and other arthropods. Chemically, they derive from linear
oligomers of (1 → 4)-␤-linked N-acetyl-d-glucosamine, with a variable number of free amino groups available for their biological
activity. This therefore depends on molecular weight, viscosity and
deacetylation degree of the oligomers/polymers (Fig. 5). Chitosans
induce different defence responses, i.e. Ca2+ transient peak, activation of MAP-kinases, callose apposition, oxidative burst, salicylate
pathway and HR, ABA synthesis, jasmonate, phytoalexins and PRproteins [122,123]. The pattern of these defence responses depends
both on the plant hosts and the physical–chemical properties
of applied chitosans, i.e. MW, deacetylation degree and viscosity
The capacity of inducing resistance against viral diseases by chitosans is long known and has been reviewed in [125]. Chitosans
induce resistance against a number of systemic viruses infecting
bean, pea, tobacco and tomato, such as bean common mosaic virus
(BCMV), bean yellow mosaic virus (BYMV), alfalfa mosaic virus
(AMV), peanut stunt virus (PSV), TMV, ToMV, PVX, PVY [125]. In
all the above-mentioned cases, the induction phase was shorter
than that of BTH, ranging from 24 to 72 h. Much efforts have been
expended to understand the mechanisms underlying this antiviral activity. Chitosan foliar sprays induced ABA-mediated callose
deposition in bean and tobacco [35] as well as a network of localized
micro-oxidative bursts, leading to micro HR and SAR against TBSV
and TNV [126]. The network of chitosan-induced micro HR would
actually mimic the effects of a pre-inoculation with an HR-inducing
pathogen (Fig. 6). Therefore, chitosan antiviral activity, although
driven by an oxidative burst as in the case of BTH, is performed in
a different way, as summarized in Fig. 7.
Unfortunately, the use of chitosans in crop protection is still
hampered by some limitations, first of all their species-specific
activity. This means that each crop may respond in a different
way and with different phytotoxicity thresholds. Thus, a careful
selection of the most suitable chitosan, both in terms of MW range
and concentration, must be preliminarily performed. Another limitation is the solubility of these products, though water soluble
chitosans (e.g. ChitoplantTM , ChitogelTM , ElexaTM , BiochikolTM ) are
now available. In any case, a question to be considered is that
chemical processing to obtain water soluble products includes
conversion of chitosan cationic oligomers to salts (e.g. to lactate,
ascorbate, acetate, etc.). This process may alter the conformation
of the chitosan polymer, generally an extended twofold helical
structure. These commercial products may show activity against
bacterial and fungal diseases, but they may not act as pathogenassociated molecular patterns, in principle a critical condition for
the induction of resistance against viruses. The most common
commercially available chitosans have MW over 10 kDa up to over
300 kDa (Fig. 5), and they must be dissolved at the concentration
of 0.01–0.2%, depending on the plant species, in a solvent, usually
diluted acetic, ascorbic, or lactic acid. Oligomers with a homogeneous composition in terms of monomer number (i.e. 6–10) are
instead water-soluble and biologically more active than polymers
[124], particularly for inducing resistance against viruses through
the transient Ca2+ influx [127]. However, they are also more expensive (per full dose), laborious to prepare and they must be used at
lower concentrations to avoid serious phytotoxicity. In this view,
low molecular weight polymers might represent an acceptable
compromise, at least for field treatments. Finally, even if the solvent
has little influence on chitosans activity as resistance inducers, it
may be determinant in impairing virus transmission by insect vectors as observed in preliminary experiments with the pathosystem
TYLCSV-tomato. In these experiments transmission of TYLCSV by
B. tabaci was significantly lowered by pre-treatment with chitosan
dissolved in ascorbic acid, but not in acetic acid, yet both solvents
alone were unable to influence transmission (Faoro and Caciagli,
In conclusion, the antiviral activity of chitosans has been demonstrated mainly in glasshouse experiments. Their use in open fields
is still a promising strategy that needs further investigation with
suitable chitosans, in terms of polymer fragment length, MW range
and crop-specific dilution. Nevertheless, some good results have
already been obtained using chitosan in combination with root
bacterization by PGPR, as described ahead. Last, but not the least,
chitosans are also antitranspirant products [128] and may also
reduce mycotoxin contamination [129], two appreciable “side”
effects in the context of a climate global change scenario and of
a strong request of healthy and safe food for everyone.
3.3. Other potentially effective compounds
In the last two decades a number of molecular structures from
diverse chemical classes and natural products have been reported
to induce systemic resistance against virus disease, as summarized
in Table 2 [130–142] and Fig. 4. Unfortunately, the antiviral activity
of these compounds has been often assessed with incompatible
virus–host combinations causing HR, thus their efficacy against
systemic infections is not known. When tested with compatible
viruses their effectiveness was usually lower than that of BTH, used
as reference compound.
A brief reference to the most known compounds is given in
Table 2.
3.4. Plant-growth-promoting rhizobacteria (PGPR) and other
beneficial microorganisms
PGPR, besides their beneficial effects on plant growth are
able to trigger a jasmonate-ethylene mediated ISR systemic
F. Faoro, F. Gozzo / Plant Science 234 (2015) 1–13
Table 2
Potential elicitors of induced resistance to viral diseases.
Compounds or extracts
Host plant
Infection type
Probenazole and its metabolite BIT
N. tabacum cv. Xanthi-nc
N. tabacum cv. Xanthi-nc
N. tabacum cv. Xanthi-nc
N. tabacum cv. Xanthi-nc
N. tabacum
N. glutinosa
N. tabacum cv. Xanthi-nc
N. tabacum
N. tabacum
N. tabacum
N. glutinosa
A. thaliana
Harpin popW
Esterified whey prot. (Me-Lactoferrin)
Spermine and longer
p-Aminobenzoic acid (PABA)
Capsicum annuum
Solanum lycopersicon
N. tabacum L.
CMV strain
B2 CMV strain I
BABA, ␤-aminobutyric acid; TMV, tobacco mosaic virus; TRSV, tobacco ringspot virus; CMV, cucumber mosaic virus; TYLC, tomato yellow leafcurl virus.
resistance. Though ISR signaling is different from SAR, the induced
resistance is similar, as it confers protection against a broad spectrum of pathogens, including viruses [17]. As mentioned above,
the exploitation of ISR against systemic virus infections has not
received much attention in the past, possibly because there are
contrasting indications of its efficacy against viruses [20]. Nevertheless, a number of studies [143] have been carried out in the last
two decades showing that containment of CMV infection is possible in cucumber and tomato cultures growing in greenhouse by
bacterization with Pseudomonas fluorescens and Serratia marcescens
[16,144]. Again, other bacteria species (Bacillus pumilus, Bacillus
amyloliquefaciens, Bacillus subtilis, Kluyvera cryocrescens) that were
selected in greenhouse experiments as resistance inducers to CMV
in tomato, were also effective in field trials [145]. Furthermore,
PGPR-treated plants were significantly taller with higher yield than
untreated controls. Tomato bacterization can be performed by seed
coating or by soil drench of seedlings before transplanting. Seed
coating is also effective in reducing the infection by tomato mottle virus (ToMoV), a whitefly transmitted virus, under high level of
disease pressure [146]. Protection against ToMoV remained for 40
days after transplantation, but no protection was observed by 80
As a rule, mixtures of PGPR strains offer more consistent and
broader spectrum protection than single strains. This is also true
in the case of CMV, where combinations of PGPR strains protected
against the virus and promoted plant growth [147]. Combinations
of PGPR and chitosan resulted in a notable plant growth promotion
[148], besides reducing CMV infection. PGPR plus chitosan treated
tomato plants, inoculated at an early developmental stage, when
their size was similar to untreated controls, were not protected
against CMV [149]. This would mean that PGPR plus chitosan confers a resistance more similar to mature plants but not induced
systemic resistance. Nevertheless, the accelerated growth is “per
se” a form of resistance because it shortens the time in which
plantlets are small and very susceptible to infection [150]. Chitosan
in combination with PGPR (Pseudomonas sp.) efficiently reduced
the infection by tomato leaf curl virus (ToLCV) in tomato fields by
80–90%, increasing considerably biomass and fruit yield [151].
Isolates of the plant growth-promoting fungus Fusarium equiseti
(GF18-3), in combination with the arbuscular mycorrhizal fungus
Glomus mosseae (Gm), have been used to study the effects of their
single or dual inoculation in cucumber plants on both plant growth
and systemic resistance against CMV [152]. The effects were compared with those obtained by a BTH soil drenching. While the
chemical application reduced plant growth with respect to the control, a beneficial effect by the dual inoculation was evident up to
7 weeks after planting and was similar to the effect afforded by the
inoculation with GF18-3 alone. The beneficial effects in reduction of
CMV disease severity were similar for both the bio-control and the
chemical treatment. In terms of the CMV concentrations measured
in leaves by ELISA, the combined inoculation GF18-3 + Gm afforded
a value of 0.54 and BTH a value of 0.51 vs. a value of 0.85 observed
in the control at 21 days post infection.
The induction of systemic resistance to CMV by pretreating Arabidopsis and tobacco plants with Penicillium simplicissimum was
tested using a barley grain inoculum of the isolate GP17-2 or its
culture filtrate [153]. The effects of partial reduction of disease
severity were similar, even if not identical, to those obtained with
BTH, but the growth effects on fresh and dry weight of Arabidopsis
and tobacco were significantly higher than the control while those
observed in BTH treatments were slightly lower than the control
itself. Bioassay data showed that both inoculum and filtrate treatments increased the expression of genes inducible by salicylate as
well as by jasmonate/ethylene, clearly indicating their capacity of
inducing systemic resistance to CMV. Interestingly, the elicitation
of ISR was not compromised in NahG transgenic plants (uncapable
to accumulate salicylic acid) of Arabidopsis, nor in mutants impaired
in the jasmonate/ethylene signaling pathways.
Systemic resistance in tobacco against TMV was induced under
greenhouse conditions by treating plants with strains of B. pumilus
(EN16) and B. subtilis (SW1) [154]. Maximum protection was
observed with 5–7 days intervals between treatment and challenge
inoculation. Disease severity was reduced by half with EN16 and
by 71% with SW1 at 14 days after challenge inoculation. A mixture of the same bacteria species used to bacterize cowpea seeds
reduced the level of infection by BCMV by 62%, in terms of virus concentration, suggesting that seed bacterization with PGPR can be a
useful method of inducing resistance to viruses [155]. This has also
been confirmed recently by inducing systemic resistance to CMV
in cucumber with a number of bacteria and the fungus Trichoderma
harzianum [156]. However, it must be pointed out that such ranges
of disease reduction are not enough to allow a large scale application in the field, particularly for those viruses, as BCMV, which are
transmitted by many aphid species and by seed as well.
4. Concluding remarks and perspectives
To control a peculiar pathogen like a virus which requires
establishing an intimate relationship with its host for a successful infection, certainly the best approach is the genetic one, by
cultivating resistant plants. Unfortunately, the availability of such
plants for cultivated crops is rare. Even when they are available,
F. Faoro, F. Gozzo / Plant Science 234 (2015) 1–13
their resistance, often relies on gene x gene interaction, which can
be easily overcome by new virus strains [157]. In this scenario,
the possibility of inducing systemic resistance with chemicals or
PGPR can be an alternative or complementary approach to manage the almost inevitable coexistence of viruses with crops. Being
not direct inhibitors of any vital function of pathogens, including viruses, chemical inducers are generally considered less liable
of producing resistant strains. The SAR-mimicked approach, is, by
itself, hardly exposed to the risk of being nullified by evolving resistant strains, for the mere reason that it depends on several genes. So,
the approach is, in principle, devoid of the risks that are weakening
other approaches, for diverse reasons banned or severely restricted.
To answer the question in the title, modulation of virus virulence
has been shown to be feasible, but so far it does not imply a total
prevention of symptoms by systemic viral diseases in open field.
The efficacy of the related treatments in the field remains as the
most important question. Though the induction of systemic resistance has proved to be effective in containing virus diseases only to
a certain extent, far from conferring full protection, this should not
be understated. In fact, also the delay of the spreading of a virus into
the crop, may be of fundamental importance in reducing yield loss,
as plants are allowed to mature at a stage of development when
they will essentially tolerate the infection [150]. Moreover, a holistic approach that integrates cultivation practices such as planting
dates to avoid vector migrations, different mulches to discourage
vectors and treatments with resistance inducers may further contain infection, as the case of TSWV in tomato reported above [112].
In this approach it must also be considered that induced resistance
is a host response and, as such, is greatly influenced by genotype
and environment. Therefore, in order to maximize the efficacy of
elicitors, particular attention should be paid to the selection of cultivars able to activate stronger level of induced resistance in a specific
environment [158]. Another issue that should be addressed is the
fitness cost of induced resistance that is to say the allocation costs
arising from the diversion of metabolites and energy away from
fitness-relevant processes such as growth and reproduction toward
defense [159]. However, in the case of virus disease the “costly
business” of plant defense [160] could be a reasonable price to be
paid in the lack of alternative treatments, except for insecticide
applications to control vectors.
Finally, there is a need for more effective resistance inducers
against virus disease, and the knowledge obtained in the last decade
on SAR/ISR mechanisms and RNA silencing, and the interactions
between them, may lead to new molecules more adequate to this
If there is a general agreement to pursue this goal, more
resources in well oriented synthetic chemistry and modes of action,
including concepts of chemical genetics, must definitely be supplied than in the past. Experience gained so far with the few
chemical inducers available showed that the results on passing
from controlled conditions (household) to the open field are often
deluding. A progress in this area of uncertainty requires an insight
on the effects that environmental stresses may have on the priming
and the resistance mechanisms triggered upon pathogen challenge.
This research was partially funded by Università degli Studi di
Milano, PUR 2009 (5197618), and partially by CNR (DG:RSTL.107),
National Council of Italy, Agrifood Department, Rome, Italy.
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