ABSTRACT

Fusarium graminearum
is one of the hemiboitrophic fungal pathogens, completing its life cycle in two
phases; a biotrophic mode of living in the initial stage and a necrotrophic
mode in the later stages of infection. Fusarium is a cosmopolitan genus of the
homothallic filamentous ascomycete. It is responsible
for causing Fusarium head blight
(FHB) or ‘Scab’ on the wheat (Triticum
spp.), barely, rice, oats and ‘Ear rot’ disease on maize. Fusarium
pathogens reduce yield and grain quality by producing trichothecene mycotoxins,
such as deoxynevalenol (DON),
nevalenol, zearalenone T-2 toxins, HT-2 toxin, diacetoxyscripenol etc during
infection by acting as the virulence factors. DON is produced during both Fusarium head blight (FHB) and Crown root
rot (CR) caused by Fusarium graminearum and
F. pseudograminearum and reported to
aid in fungal colonization of host tissues. These Trichothecenes inhibit
protein synthesis by binding to the 60S ribosomal subunit subsequently activating
cell signaling program that results in apoptosis. DON being one of the
important mycotoxins is proposed to act in two ways; at lower concentrations it
indirectly stops the damage and inhibits the cell death but when produced in
higher amounts it induces the cell death in wheat. It has been suggested that
DON production levels may affect the cellular responses in a way that could
either promote necrotrophic fungal growth by initiating programmed cell death (PCD)
or reduce fungal growth by triggering antimicrobial protein accumulation and
defense gene expressions. Further, mycotoxins are also implicated to play a
role in virulence and pathogenesis on wheat.  The present study will focus on the review of literature
highlighting impact of Fusarium
graminearum mycotoxins in cell death-triggered defense responses of wheat (Triticum aestivum) and its future
perspectives.

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Keyword:Deoxynevalenol,
Hemibiotrophic, Fusarium graminearum.
Triticum aestivum, apoptosis.

INTRODUCTION

Plants being sessile are
unable to move from one place to another, so they are as usual exposed to
number of the external environmental influences. So to give an effective response
to external signal whether in the form of abiotic (temperature changes,
salinity, drought etc or biotic stress such as the pathogens in the form of
bacteria or filamentous fungi, have developed a multitude of defence mechanisms.
In response to pathogen, plants have developed strong immune signaling pathways
and mechanism that successful pathogens have evolved to escape or suppress. One
 of the basic layer of defence is the conserved
pathogen molecule (PAMPs) recognition from the plant side by the assistance of
the pattern recognition receptors (PRRs) present on the cell surface which
results in the PAMP or pattern triggered immunity (PTI),but at the same time it
has been found out that, PTI can be suppressed by the protein molecules
commonly known as the ‘effector proteins’ that are delivered either into the
cell cytoplasm or in the apoplast and they finally result in effector triggered
susceptibility (ETS).the second type of defence is the     effector
triggered immunity in which there is an interaction between the R and the
corresponding Avr gene. Hypersensitive response (HR) being an important
hallmark of the ETI which results in the programmed cell death at the sites of
infection. Fusarium graminearum is a
filamentous pathogenic fungi that can infect large no of cereal grains like
barley, wheat, corn and oats by causing the destructive disease Fusarium head blight (FHB) which results
in the reduced grain quality and production The initiation of disease starts by
deposition of the fungal spores on or inside the spikelets (Bushnell et al.,
2003).on the exterior surfaces of the florets and glumes, hyphal development
takes place rather than by direct insertion via the epidermis, prior to the colonization
of anthers, stigma and lodicules(Bushnell et al., 2003).In wheat  fungus spreads from spikelet to spikelet via
the vascular tissue in the rachis and rachilla(Trail, 2009) and this is
associated with the production of the virulence factor Deoxynivalenol that
causes necrosis of the tissues (Jansen et al., 2005).but in other cereals like
barley it has been shown that virulence does not appear  because of the toxins and the spread of
disease is limited(Maier et al., 2006).

Historical
background:

The disease was first
reported in 1884 from England where it was known as the wheat scab but later on
it spread to other humid and semi-humid areas of world where wheat is grown
especially Canada, USA,Europe, Asia, South, America (Dickson, 1942). However fusarium
head blight of wheat and barley in India was of mare importance. But in
northern parts of country it became a major constraint in the wheat production.
Almost from 1990’s, FHB appeared in epidemic from4-5 times in different parts
of Punjab like in Gurdaspur district. This fungus not only affects the host
plants, but also poses a risk problems to the animals and humans and
contaminates food and the feed products as well by the help of no of mycotoxins
which are mainly Trichothecenes (reviewed by Goswami and Kistler 2004; Xu and
Nicholson 2009).It is considered as one of the most important
“Hemiboitrophic”pathogens as it starts its pathogenicity cycle by growing
subcuticularly and intracellularly  for a
period of time as a “biotrophic” and then enters into the” necrotrophic” stage
by causing death of the particular tissue and then takes the nutrition form
that (Brown et al. 2010; Walter et al. 2010).during anthesis ,warm and wet
weather  adds to the infection and
reduces the grain yield and quality.

Fusarium graminearum genome:

Fusarium
graminearum being a homothallic does not need two
parents, it has both the sexual as well as the asexual lifecycle (Mathre
1997; Parry et al. 1995).Due to the devastating effects of the Fusarium graminearum it has been under
intensive investigation for no of years to understand its genetic basis of the
life cycle, population biology and pathogenicity and evolution.    The genome of the Fusarium graminearum isolate has been completely sequenced about 15
years ago (Cuomo et al. 2007). It has been shown that Fusarium graminearum genome is compartmentalized into the different
regions which are responsible for the essential functions and for the
specialized virulence of the host .These regions have been referred as the core
genome and the accessory genome. Broad institute has provided the most recent
annotation in the assembly 3 sequence showing almost 13,321 proteins. So the
availability of the full genome sequence revitalizes the gene function research
in F.graminearum (Ma et al., 2010). This fungus is currently posing serious
agricultural problems in several parts of the world. The infection starts in the
spikelet during the early stages of flower development which results in
shriveled grain, bleaching (Fig.1.) whereas the indirect impact results in the
contamination of grain with

Fig.1. Symptoms of Fusarium head blight on
the external surface of wheat ear glumes

                           A
Champeil, June 2004,

mycotoxins. These mycotoxins
lead to various socio economic impacts as they result in the quantitative as
well as the qualitative losses to the wheat and barley causing about 35-61%
reduction in the yield. In 2010, Pestka have fully reviewed the mechanisms of
action and the toxicological relevance of toxins.

Production
of Mycotoxins:

Deoxynevalenol (DON) is a
trichothecene mycotoxin, also known as the vomitoxin produced by the Fusarium graminearum (sexual stage:
Gibberella zeae) and Fusarium culmorum
(Miller, 1994; Desjardins, 2006).It is the major mycotoxin produced by the Fusarium graminearum It is responsible
for the emesis and refusal of feed in animals (Forsyth et al., 1997).It affects
the quality and production of grains and the health hazards associated with the
DON contaminated food includes various immunotoxic and neurotoxic effects in
animals (D’Mello et al., 1999; Desjardins, 2006).Other trichothecenes like
nivalenol (NIV) and T-2 toxin are also produced by fusarium head blight causing
pathogens (McCormick., 2003; Gale, 2003).These mycotoxins have been involved in
the pathogenesis, phytotoxicity and the induction of apoptosis. Some of the
studies have shown that there is a positive effect of nitrogen fertilizers on
the increasing incidences of the Fusarium
infected grains in wheat it is because nitrogen largely influences the water
potential of the plant and then leads to the increased susceptibility. On the
other hand a no of surveys have indicated that applying nitrogen at higher
levels than the recommended doses may reduce the head blight in the winter
wheat (Teich and Nelson, 1984).These trichothecene mycotoxins act as the
virulence factors and exerts multiple effects on the eukaryotic cell functions
and primarily inhibiting the protein synthesis.DON is considered as one of the
least toxic mycotoxin as compared to other trichothecenes.these mycotoxins
sometimes appear in conjugated forms such as recently identified glycosylated
derivatives of DON and ZEA.To counter act the action of these microbial toxins
and other xenobiotic,mechanisims including their detoxification pathways by
conjugation to the endogenous metabolites like the process of glycosylation,
acylation, conjugation  with different
amino acids and the most dedicated peptide Glutathion. Studies have shown that
the biotransformed products are passed to apoplast and the vacuoles. It has
been first reported in Arabidopsis
thaliana that the most probable detoxification reaction to detoxify the DON
in planta is its conjugation with the sugar molecules like glucose
(Poppenberger b et al., 2003). In addition to the Arabidopsis thaliana, it was also reported from the Fusarium graminearum treated wheat. New
and advanced techniques are being used like the liquid chromatography LC
associated with the mass spectroscopy MS for the detection of the
biotransformation products.  

Fig.2. LC-HRMS/MS spectra of the DON biotransformation
products.

PLOS ONE 10(3):e0119656

Biotransformation of
mycotoxin deoxynevalenol in Fusarium
susceptible and resistant near isogenic wheat lines have been well studied by (Bernhard
et al in 2015).where they have clearly characterized and identified them by
liquid chromatography-high resolution mass spectroscopy (LC-HRMS).They have
suggested that there are two major pathways one is glycosylation and the other
as Glutathion conjugation where by DON is conjugated to the different
endogenous metabolites. In addition to this they have also found the putative
sugar alcohol (hexitol) one of the DON conjugate. Some of the wheat cultivars
are more resistant to the spikelet bleaching induced by the DON and then covert
this more toxic mycotoxin into the less toxic conjugate D3G (Lemmens et al,
2005).this resistance to spread of FHB traits have been due to the presence of
the quantitative trait locus on the short arm of chromosome 3 (QTL Fhb1) and this study has also found out
that the Fhb1 wheat lines show
elevated D3G/DON ratios.FHB resistance is an important approach shown by some
of the resistant varieties which appears as the most important aspect and
protects the wheat from the disease, so far as the breeding programmes are
concerned the effective resistance genes are lacking(Bai and Shaner.,1996;Rudd
et al.,2001).Sumai 3 ,a Chinese cultivar exhibits the resistance type second
and is considered as the most efficient  source of resistance (Anderson et
al.,2001).Buerstmayr and co workers in 
2003 have recognized the Quantitative trait loci (QTL) that is involved
in conferring both type ?  and type
??   resistance.

DON
and its role in eliciting defence gene responses in host:

Deoxynevalenol is one of
the sesquiterpeniod epoxide trichothecene responsible for the induction of the
defence response at the higher concentrations.DON localization occurs in the cytoplasm,
plasma lemma and the chloroplast, sometimes it has been found in the ER and
ribosome also (Kang and Buchenaur 1999) this mycotoxin also alters the membrane
permeability of the cells (Bushnell and Seeland 2006) It has been proved that
DON binds with the 60s ribosome and there by inhibits the eukaryotic protein synthesis
at concentrations well below the 100 mg/l (Nishiuchi et al., 2006). The Maximum
acceptable level of Deoxynivalenol in the grains of wheat ranges from 0.5 to 2
mg/kg in USA, Canada, and some other Europeans countries (Snijiders 1990).In
wheat infected grains, the levels of DON vary significantly (Miller et al.1985,
Mirocha et al.1994, Liu et al.1997).The levels of DON in susceptible cultivars
were reported as eight fold higher than in the resistant cultivar (Miller et
al.1985). To prove this different analysis like western blots were done to
asses protein levels on 100 mg/l DON treated wheat samples using PR2 and PR3 (ß-1,
3-glucanase and chitinase) specific antibodies. Earlier studies have shown that
DON production by Fusarium plays an
important role in the fungal colonization of host tissues during both Fusarium head blight (FHB)  and crown rot (CR) caused by the pathogen
(Jansen et al.,2005;Langevin et al.,2004; Maier et al.,2006;Mudge et
al.,2006).previous studies done by (Olivia j. Desmond et al., 2008) have
indicated that a range of  defence responses
are induced by the said mycotoxin which includes the most and basic response
that’s ROS production which may be partially responsible for the defence gene
transcripts ,their protein products and programmed cell death.Oxidatiive stress
in wheat cells is caused by the DON and DON producers (Zhou et al.2005;Golkari
et al.2007;Desmond et al.2008).One study shows that ROS generation and
Cytotoxicity are the mechanisms of mycotoxin mediated toxicity.ROS are highly
reactive due to the presence of the unpaired electrons so are very much
reactive. These have important role in the signaling and homeostasis of the
cell. Under the conditions of normalcy, they are cleared from the cell by the
action of the antioxidants like superoxide dismutase (SOD), catalase and
glutathion peroxidase (GPx). This indicates that mycotoxin DON may be assisting
the necrotrophic growth of the fungi by inducing death of the host plant.
During the FHB and crown rot of host, the rate and extent of the disease is
influenced by the contrasting effects of the DON. It also stimulates the
antimicrobial defence responses in the wheat. Even the model plant Arabidopsis
has been subjected to the rang of the trichothecene including the T-2toxin, HT-2
toxin, diacetoxyscripenol and DON and has shown that all these toxins can cause
induction of defence gene transcripts and the cell death, also the protein
synthesis in Arabidopsis cell suspension showed 50%inhibition by 1.5mg/L DON (Masuda
et al., 2007; Nishiuchi et al., 2006).they have further suggested that DON may
be acting as a protein synthesis inhibitor at concentrations below the
threshold required to activate Arabidopsis
defence responses (Nishiuchi et al.,2006). Some of the up regulated genes at
transcriptional level were found out to be involved in the inactivation of the
brassinosteriods, programmed cell death related proteins and transcription factors,
transport and trichothecene detoxification proteins, ubiquitination related
proteins, and cytochrome P450 (Ansari et al,2007;Buddo et al,2007;Masuda et
al,2007;Desmond et al,2008).Stephanie W et al have shown that the wheat
spikelet ability to resist the bleaching caused by the deoxynivalenol is
genotype dependent trait (Stephane.W et al 2008).Germin-like and the peroxidase
genes were fund out to be the most highly transcriptionally induced gene
following the DON treatment as these are related to the metabolism of the
reactive oxygen species (Liuet al.,2005;Zimmermann et al., 2006).  As these functional observations were also
identified in the wheat plants during infiltration of DON and are consistent
with the ROS production. It has been suggested that the application of the
ascorbate an antioxidant along with the DON markedly reduces the level of
transcriptional activation of the peroxidase genes by DON; however the actual
effects of antioxidant (ascorbic acid) on the activity of DON are still
unknown. Earlier research done on the mycotoxins of Fusarium have demonstrated very well that DON causes the cell death
in wheat and is associated with the laddering of the genomic DNA ,which is a
strong hallmark of the programmed cell death in the plants as well as other
eukaryotes (Ryerson and Heath,1996;Tada et al., 2001). PCD is an active process
which helps in the removal of the unnecessary cells from the body as is one of
the protective mechanisms in plants to get rid of the worn out cells and
tissues. So this active process of cell death requires the de nova synthesis of
proteins (Tada et al., 2001).Further it was shown that the eukaryotic protein
synthesis inhibitor cyclohexamide along with deoxynevalenol prevents the cell
death 24 h after treatment. However the mechanisms behind these protective
functions are unknown, although it may be possibly due to the synthesis of the
specific protein by cyclohexamide that might be involved in the inhibition of
the DON induced cell death in the wheat. During PCD which is induced by H2O2
in plant cells which acts as one of the important signaling molecule by
stimulating PCD (Houot et al., 2001; Yoda et al., 2006) also it was found out
that DON infiltration along with the ascorbic acid leads to the decrease in the
genomic DNA laddering and also cell death. In comparison to the F.graminearum, other species of fusarium
were also reported to induce cell death by the depletion of extracellular ATP
(Chivasa et al., 2005).During the stimulation of cell death by mycotoxins,
nutrient leakage is one of the main problems, facilitating the necrotrophic
fungal colonization i.e. growth and spread throughout the host. Necrotrophic pathogens
also produce toxins which induce cell death. This sort of functional
characterization of DON is consistent with its DON-nonproducing mutant Fusarium to infect wheat spikelets which
surrounds the point of infection during the Fusarium
head blight FHB (Jansen et al., 2005).During the crown rot disease in wheat it
was found that relatively higher concentrations of DON causes the induction of
host cell death and H2O2.However this higher
concentrations of DON occurred in the later stages of the disease when visible
lesions develop in the host. Data presented by one of the studies have added to
the fact that trichothecenes play important roles in the fusarium pathogenesis.
The roles of trichothecenes have became apparent from the UV mutagenesis and
gene disruption studies that attributes two features first, trichothecenes
appear to exhibit some degree of host specificity. That is the inability to
produce these mycotoxins is associated with reduction in virulence on some
hosts but unable to affect virulence on other hosts (Desjardins et al.1992;
Adams and Hart 1989). The Tri5 gene
disruption leads to inability to produce trichothecenes and also leads to the
physiological changes. It is also possible that the inability of Tri5 mutant to produce
trichodiene-derived compounds other than trichothecene caused reduction in
virulence of GZT26 and GZT40 (Greenhalgh et al.1989).trichothecene deficient
mutants were created by gene disruption to check whether trichothecene
production contributes to the virulence of F.
graminearum. The disrupted gene Tri5
encodes the enzyme trichodiene synthase that is involved in the trichothecene biosynthesis
(Proctor et al. 1995). Since their s no single solution to the FHB in wheat, so
the fully integrated and currently available tools are mandatory and
incremental improvements are needed in all fronts (Schaafsma et al., 2005).Zearalenone
is the other mycotoxin produced by the F.graminearum
in wheat and other cereal crops. In animals it leads to the estrogenic effects.
It binds to the ER as an agonist. Phytohormones are reported to play a major
role in the signaling pathways and defence responses in the host.Salicyclic
acid (SA), Jasmonic acid (JA), methyl jasmonate (MeJA), abscisic acid (ABA) are
identified as the regulators of the wheat Fusarium head blight. It was found
that the wheat ABC transporter which is involved in the pleiotropic drug
resistance (TaPDR7)  is negatively
regulated by these hormones except the 
indole acetic acid (IAA) which is involved in the positive regulation of
this ABC transporter (Wang.G et al 2016). SA plays an important role in the
resistance of wheat against the F.graminearum
(Mona Sorahinober et al., 2015).Different inoculation assays have been tried
for the screening of the FHB in wheat, one of the easy, rapid and reliable
assay developed so far is the clip-dipping seed or seedling inoculation   method which helps us to compare the various
levels of the resistance in different varieties of the wheat (Sanghyun Shin et
al., 2013)

Conclusion
and Future perspectives

Earlier investigations
have very well demonstrated that the action of mycotoxin DON is appreciating
the disease spread in wheat from one spikelet to another via the intermodal
rachis (Carin Jansen et al., 2005). On the other hand, recent studies done on
the Fusarium in wheat have shown that
without the trichothecene production, wheat stops the movement of the fungus by
strong cell wall fortifications in the rachis nodes. Trichothecenes are the one
strong helpers of the Fusarium in
inhibiting this defence response in wheat and helps in the ccolonisation.if the
infection occurs via the epicarp, it will lead to the early cell death and by
sure the vigorous and rapid fungal growth independent of the trichothecene
production. More approaches are needed and suggested, as it is evident from
different investigation done on the F.graminearum
secretion of different cell wall degrading         enzymes like cellulose of host cell wall,
xylans and pectins etc (Kang et al., 2000; Wanyoike et al., 2002). Similarly
host secretes inhibitors of these enzymes like xylanase inhibitor (XIP)
(Goesaert et al., 2003) or wheat (TAXI) (Juge et al., 2004) might help in
preventing the fungus to enter the cell via the fruit coat tissues (Di Matteo
et al., 2003). To understand the defense responses in the wheat, detailed
studies needs to be done. To evaluate the strong possibilities of elimination
and targeting of the various mycotoxins involved in the Fusarium head blight
disease and elicitation of the death caused by the mycotoxins. The
breeding  programmes aims at preventing
the infection in wheat through fruit coat have been found as same in barley as
well ,still different strategies needs to be explored which will be helpful in
finding out the ways to decrease the infection spread and will improve the type
second resistance against the fungal infection in wheat. Development of the FHB
resistant wheat varieties is of course the most important in combating the
diseases and will help in reducing the agricultural and economic losses.

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