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International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Telangana, India.
Phytotoxins are low molecular weight metabolites produced by plant pathogens that cause obvious damage to plant tissues
and are known to be involved in plant disease. Phytotoxins from various formae speciales of Fusarium oxysporum such as
fusaric acid from the banana pathogen F. oxysporum f. sp. cubense, beauvericin from the muskmelon pathogen F. oxysporum f.
sp. melonis, and bikaverin and norbikaverin from the cotton pathogen, F. oxysporum f. sp. vasinfectum have been reported to
cause wilt symptoms in their host plants. Fusarium wilt, caused by Fusarium sp. one of the serious disease of chickpea, is
responsible for losses up to 100 per cent when conditions favour the disease. Chlorosis and wilting are common symptoms on the
chickpea plants infected with Fusarium sp. These symptoms suggest that phytotoxins are involved in the Fusarium wilt disease
of chickpea. Filtrates from cultures of Fusarium acutatum, caused permanent wilting of chickpea cuttings and killed cells in a
bioassay. The phytotoxin from the culture filtrate was identified as 8-O-methyl-fusarubin. Knowledge of such phytotoxic metabolites
provides insights into disease syndromes and may be exploited by conventional and molecular breeding to obtain crops
resistant to plant disease.
KEYWORDS: Phytotoxins, Fusarium wilt, chickpea, disease development
Chickpea (Cicer arietinum L) is the third most
important pulse crop after bean (Phaseolus vulgaris) and
pea (Pisum sativum) on a world basis but of first
importance in the Mediterranean basin and South Asia.
It is grown in 33 countries on an area of about 11.5 million
hectares (Bidyarani et al., 2016) and India accounts for
about 65 per cent of the world’s chickpea production
(FAOSTAT, 2014).Cultivated chickpea are divided into
two major groups “desi” and “kabuli”. Chickpea seed is
mainly used as food because of its high protein (12-31
%) and carbohydrate (52-71 %) contents (Awasthi et al.,
1991). Global yields of chickpea (968 kg ha-1) have been
relatively stagnant (FAOSTAT, 2013) for the last five
decades in spite of using various advanced and molecular
breeding approaches, extensive use of synthetic fertilizers
and pesticides that in addition created environmental and
health concerns. Productivity may be considerably
improved if the adverse effects of abiotic and biotic
stresses are reduced. The major abiotic stresses are cold,
heat and drought. Of these, drought is the major limiting
factor as chickpea is grown on residual soil moisture as a
post-rainy season crop. Advancing sowing dates in certain
regions can alleviate the effect of moisture stress and
thereby increasing the yield. Chickpea is also sensitive
to salinity and this is an important problem in India and
Pakistan. Numerous studies have shown that soil salinity
inhibits legume growth and development and decreases
nodulation and nitrogen fixation (Mensah and Ihenyen,
2009; Egamberdieva et al., 2013). Salt tolerant genotypes
are available but these lines do not generally yield well.
Lowering the water table through improved drainage can
be effective in areas where it is high (ICRISAT, 1997).
Chickpea suffers from about 172 pathogens
consisting of fungi, bacteria, viruses and nematodes of
which 38 are soil-borne. Rhizoctonia solani, Sclerotium
rolfsii and Fusarium oxysporum f. sp. Ciceri (FOC) are
the most serious and are responsible for wet root rot, collar
rot and wilt, respectively, and cause losses as high as 60
to 70 per cent when conditions favour disease (Anjaiah
et al., 2003). The foliar diseases which may damage
chickpea are blight caused by Ascochyta rabiei and grey
mould caused by Botrytis cinerea. Bacterial blight caused
by Xanthomonas cassiae was also found damaging in
India (Nene, 1980). Important viral diseases include stunt,
chlorosis and dwarfing, mosaic, proliferation and necrosis
caused by Pea Leaf Roll Virus, Chickpea Chlorotic Dwarf
Virus, Alfalfa Mosaic Virus, Cucumber Mosaic Virus and
Lettuce Necrotic Yellow Virus, respectively (Horn and
Reddy, 1996). Among the nematodes, Meloidogyne spp.,
Heterodera spp. and Pratylenchulus spp. cause heavy
losses of the crop in several countries (Ansari et al., 2002).
Fusarium wilt and Ascochyta blight are considered to be
the two most devastating diseases of chickpea (Hamid et
al., 2001).
Fusarium wilt of chickpea
Fusarium oxysporum is the causal agent of wilt of
many plant species. All strains may exist saprophytically
and some are considered to be non-pathogenic but many
are well known for inducing wilt on a variety of plants
(Fravel et al., 2003). Often isolates are specific to particular
hosts for example, F. oxysporum f. sp. lycopersici infects
tomato and F. oxysporum f. sp. cubense infects banana
(Fravel et al., 2003).Wilt of chickpea is normally
considered to be caused by F. oxysporum f. sp. ciceri
(Padwick) Snyd. and Hans, hereafter designated as FOC.
Initially it was believed that formae speciales were
specific to one host and hence the name was taken from
the host. However, other species and formae speciales of
Fusarium also cause wilt in chickpea (Di Pietro et al.,
2003; Gopalakrishnan and Strange, 2005). Fusarium wilt
is prevalent in all chickpea-growing areas of the world,
including India, Pakistan, Spain, Iran and Tunisiaand is
important where the chickpea-growing season is dry and
warm (Dubey et al., 2010). This disease causes yield
losses up to 100 per cent under favorable conditions in
chickpea (Anjaiah et al., 2003; Landa et al., 2004).
Symptoms of Fusarium wilt in chickpea consist of
epinasty, chlorosis of leaves, discoloration of vascular
tissue and ultimately collapse of the whole plant (Hamid
et al., 2001). The disease may be diagnosed by sudden
drooping of leaves and petioles, which may turn yellow
and browning of vascular bundles and its colonisation by
fungal hyphae, which are apparent when the stem is split
open (ICRISAT, 1995). Seven races of FOC (0, 1, 2, 3, 4,
5 and 6) have been reported worldwide (Cachinero et al.,
2002).
Management of Fusarium wilt
FOC may be eliminated from the seed using the
fungicide Benlate T (30% Benomyl + 30% Thiram) at
0.25% (Mandeel, 1996). FOC can survive in the soil for
more than 6 years and also in symptomless carriers
(Haware and Nene, 1982). Therefore it is not possible to
control the disease by normal crop rotation. Soil
solarisation reduced FOC population and incidence of
wilt (Chauhan et al., 1988) however; cost considerations
would limit the use of this technique in the commercial
farming. Sterilisation of the soil by methyl bromide is
not an option as it is both costly and environmentally
damaging (Fravel et al., 2003). Date of sowing seems to
have an effect on the incidence of wilt by lowering the
fungal attack but also yield.
Disease resistance is another way to control plant
disease if satisfactory levels of long-lasting resistance can
be incorporated into culturally desirable crop plants.
Maintenance of high levels of resistance to disease is
normally achieved by selection and hybridisation.
Selection involves exposing plant populations to high
disease pressure and selecting individuals that survive.
Resistance can be also developed by mutagenesis using
chemicals such as methyl or ethyl-methanesulphonate,
diethyl sulphate or ionising radiation such as X or gamma
rays. Generation of resistance by gamma radiation has
been reported for diseases including wilt, blight, stunt
and root rot of chickpea but none of these has yet reached
commercial application. Although varieties of plants that
are resistant to some fusarial diseases are known, e.g.,
tomato grown in greenhouses are resistant to common
races of F. oxysporum f. sp. lycopersici (Fravel et al.,
2003), but there are several plants for which for no
dominant gene for disease resistance to Fusarium is
known e.g. carnation, cyclamen and flax. Despite the
presence of races of the fungus, chickpea in relation to
FOC appears to fall into this category. Several workers
have observed different patterns in the development of
wilting symptoms when chickpea is infected with FOC.
For e.g., Sharma et al. (2012) identified moderate level
of resistance against Fusarium wilt on three breeding lines
(ICCV 05527, ICCV 05528 and ICCV 96818) and one
germplasm accession (ICC 11322).
Biological control of plant diseases usually occurs
by one or more of several distinct mechanisms. These
include competition for nutrients, parasitism, antibiotics
production and induced systemic resistance (Van Loon
et al.,1998). Biological control of the soil and seed-borne
plant pathogenic fungi have been addressed using
bacterial and fungal antagonists, to certain extent. Strains
of Bacillus spp., Pseudomonas spp. Trichoderma spp. and
non-pathogenic isolates of F. oxysporum were found not
only to control FOC but also in helping the chickpea plants
to mobilize and acquire nutrients (Postma et al., 2003;
Perner et al., 2006; Gopalakrishnan et al., 2015).
Saprophytic Fusarium are able to suppress populations
of pathogenic Fusarium spp. by competing for nutrients
in the soil, infection sites on the root and also in inducing
systemic resistance (Fravel et al., 2003). In wilt sick plot,
Landa et al. (2004) reported that biocontrol agents,
Bacillus subtilis GB02 and Pseudomonas fluorescens
RG26, when applied alone and in combination with nonpathogenic
F. oxysporum Fo 90105 delayed the disease
onset and suppressed Fusarium wilt.Trichoderma
harzianam and Pochonia chlamydosporia were found
effectively controlled Fusarium wilt in chickpea (Khan
et al., 2011). Streptomyces spp. isolated from national
parks in Kenya were shown to have antifungal activity
against FOC (Nonoh et al., 2010).Five strains of
Streptomyces spp., isolated from herbal vermi-compost,
were reported as having potential for biocontrol of
Fusarium wilt in chickpea (Gopalakrishnan et al., 2011).
Although biological control often appears promising in
specialised environments, disappointing results frequently
are obtained in the field as several factors determine the
survival and delivery of the antagonist. Therefore, the
strategy to combat the disease should be to integrate
different methods of control including cultural practices,
use of resistant cultivars, biological control and chemical
control. For instance, Singh et al. (2003) showed that two
strains of P. fluorescens in combination with thiram @
1.5 g kg-1 effectively controlled collar rot of chickpea
caused by S. rolfsii in both greenhouse and field
experiments. There is a growing interest in the use of
secondary metabolites, such as toxins, proteins, hormones,
amino acids and antibiotics from microorganisms for the
control of plant pathogens as these are readily degradable,
highly specific and less toxic to nature (Doumbou et al.,
2001). Hence, metabolites from microorganisms may be
exploited for the control of Fusarium wilt.
Phytotoxins (metabolites) of microorganisms
Phytotoxins are low molecular weight compounds
produced by microorganisms that cause obvious damage
to plant tissues and are known with confidence to be
involved in plant disease (Scheffer, 1983). Such damage
may include wilting, water soaking, chlorosis and necrosis
(Strange, 2003). For instance, the strawberry pathotype
of Alternaria alternata produced AF toxin correlated with
the pathogenicity of the isolates (Akamatsu et al., 1997).
Coriander seeds soaked in spore suspension of F.
oxysporum f. sp. corianderi and partially purified toxins
significantly lowered the seed germination and reduced
the shoot and root lengths over the un-inoculated control
(Gandhikumar and Raguchander, 2001).
Phytotoxins have been described in a number of welldocumented
reports as integral factors in disease
development (Yoder, 1980; Scheffer, 1983) and have
proved to be useful tools in the selection of resistant/
tolerant plants (Daub, 1986).Phytotoxins may be
classified as host-selective (host specific) or non-selective
(non-specific). Host-selective phytotoxins are toxic to
those plant species or cultivars that serve as hosts for the
toxin-producing pathogen and lack toxicity towards nonhosts.
A non-selective toxin may exhibit differential
toxicity towards various plant species but toxicity is not
highly correlated with the toxin-producer’s host range
(Knoche and Duvick, 1987). Host-selective toxins are
found principally in species of Alternaria and
Cochliobolus and non-selective toxins in species of
Fusarium, Ascochyta, Leptosphaeria and also some species
of Pseudomonas and Xanthomonas (Tables 1 and 2).
Plant pathogens produce a variety of secondary
metabolites in culture that show phytotoxic activity but
only a small proportion of these have a demonstrated role
in plant disease. This is because of their low water
solubility and the extreme sensitivity of the plants to
solvents used to dissolve these compounds. Many of these
phytotoxic compounds dissolve in solvents such as
methanol, ethanol, dimethyl sulfoxide and acetone at a
concentration of two to five per cent, which are extremely
damaging to crop seedlings. These solvents upon further
dilution usually causes the compound to precipitate,
leaving a negligible concentration of the solution.
However, determination of the role of phytotoxic
compounds in pathogenesis (ability to cause disease) or
virulence (severity of disease) is critical and hence
pathogenicity studies should precede any effort to
correlate toxin production with pathogenicity and
virulence (Strange, 2007).
Purification of phytotoxins
Although phytotoxins are thought to play a role in
plant disease syndrome, particularly if the symptoms are
expressed at the site of infection, they are usually difficult
to extract from the infected plant. Phytotoxins which are
of importance in plant disease syndromes are usually
isolated from axenic cultures of pathogens. For examples,
isolates of Ascochyta rabiei, the causal agent of blight in
chickpea, produced the toxins, solanopyrones A and C
when grown in Czapek Dox liquid medium (CDLM)
supplemented with chickpea seed extract (Alam et al.,
1989) and also solanapyrone B when grown on CDLM
supplemented with metal cations, Zn, Ca, Cu, Co and Mn
(Chen and Strange, 1994). These toxins were isolated by
solvent partitioning with ethyl acetate and flash
chromatography of the organic fraction on silica gel. The
compounds were identified in the flash fractions by their
characteristic UV spectra and those with similar spectra
were combined. Purity of the compounds in the combined
fractions was monitored by HPLC on an analytical C18 column
with aqueous mixtures of methanol, acetonitrile and
tetrahydrofuran as mobile phases (Hamid and Strange, 1997).
Detection of an unknown toxin can be achieved by
a suitable bioassay; preferably the assay should be rapid
to perform, simple, sensitive and give quantitative results
(Strange, 2003). Shohet and Strange (1989) suggested a
bioassay technique in which cells were isolated from
leaves of pigeonpea by a combination of enzyme digestion
and mechanical agitation followed by incubation with
culture filtrates of Phytophthora drechsleri f. sp. cajani.
Phytotoxic compounds from P. citrophthora were assayed
with tomato (non-host) and lemon (host) seedlings
(Breiman and Barash, 1981) whereas tomato cuttings were
used to assay toxins produced by P. cactorum in culture
(Pligh and Rudnicki, 1979).
Phytotoxins from Fusarium species
Fusarium species produce complex mixtures of
toxins that probably serve a variety of functions in
allowing them to compete with other microorganisms and
dominate their habitats. Species of Fusarium are known
to produce mycotoxins, phytotoxins and some of the
toxins, such as the trichothecenes, are toxic to both
animals and plants (Desjardins et al., 1992 and 1995).
Bosch et al. (1989) reported that out of 62 isolates of
Fusarium, obtained from pasture grass and soil from New
Zealand, 82 per cent of the isolates were toxic to rats in
feeding tests and of them 24 per cent were found severely
toxic and caused haemorrhages of stomach, intestine,
haematuria and finally death. Some of the compounds
produced such as the trichotecene toxins, deoxynivalenol
(DON), T-2 toxin and diacetoxyscirpenol (DAS) as well
as zearalenone (ZEN) and the fumonisins have
mammalian toxicity (Cawood et al., 1991; Hussein and
Brasel, 2001; L’vova et al., 2003). Fumonisin mycotoxins
(FB1 and FB2) produced by the fungus Fusarium
moniliforme were extracted from the cultures of the
fungus on maize meal with methanol/water (3:1) and
further purified using Amberlite XAD-2, silica gel and
reversed phase C18 chromatography (Cawood et al., 1991).
Schaafsma et al. (1998) demonstrated a cheapest and
reliable method for identifying and quantifying DON
(produced by Fusarium graminearum and F. culmorum
in maize) and zearalenone (produced by F. graminearum
in stored grain) with thin layer chromatography. Several
toxins from various formae speciales of F. oxysporum
have been described as causing wilt symptoms in their
host plants. These toxins include fusaric acid from the
banana pathogen F oxysporum f. sp. cubense, beauvericin
from the pathogen of muskmelon F. oxysporum f. sp.
melonis, and several polyketide toxins (including
bikaverin and norbikaverin) from the cotton pathogen, F.
oxysporum f. sp. vasinfectum (Thangavelu et al., 2001;
Moretti et al., 2002; Bell et al., 2003).
Phytotoxins from Fusarium species causing wilt in
chickpea
Chlorosis and wilting are common symptoms of
toxicosis and these symptoms are characteristic of the
phenotypes of chickpea plants infected with FOC
(Gopalakrishnan and Strange, 2005). Other symptoms of
toxicosis on chickpea are epinasty of the leaves,
discoloration of the vascular tissue and ultimately collapse
of the plant (Hamid et al., 2001). These symptoms suggest
that phytotoxins are involved in the disease. Kaur et al.
(1987) found that partially purified toxin from FOC
inhibited callus growth in chickpea. Rao and Padmaja
(2000) reported that crude culture filtrates of FOC, when
diluted to 30 per cent with water, caused wilting of 1-
week-old chickpea seedlings in 4-5 days.An isolate of
FOC from Thal region of Pakistan, identified based on
its morphology and pathogenicity, was further identified
as Fusarium acutatum in the 16S rDNA analysis
(Gopalakrishnan and Strange, 2005). Filtrates from
cultures of F. acutatum grown on a defined liquid medium
caused permanent wilting of chickpea cuttings and killed
cells, isolated enzymatically from healthy plants, in a
bioassay (Gopalakrishnan et al., 2005).
Purification of thephytotoxins from the culture
filtrates of F. acutatum
Toxic activity from the culture filtrates of F. acutatum
was retained by a cyano solid phase extraction cartridge
and the toxin was isolated by elution from the cartridge
in acetonitrile and Si-gel thin layer chromatography of
the eluate. Bioassay of the fractions were done as per the
protocols of Hamid and Strange (2000). In brief, filtrates
of cultures were tested for their toxicity to cells isolated
from chickpea leaflets using fluorescein diacetate to
differentiate live and dead cells. This compound readily
enters the live cells with intact plasma-membranes and,
once inside the cell, is metabolised by esterases to give
free fluorescein. As plasma-membranes are impermeable
to fluorescein, the compound accumulates and imparts a
yellow-green fluorescence to such cells, which may be
viewed under fluorescent microscope.
Analytical HPLC of the compound on a cyano
column with diode array detection gave a single peak
with a homogenous spectrum and max D 224 and 281
nm whereas NMR and mass spectrum studies showed
that toxin was 8-O-methyl-fusarubin (Gopalakrishnan et
al., 2005). Naphthazarin toxin, produced by species of
Fusarium, of which 8-O-methyl-fusarubin is one, have
been implicated in disease syndromes in citrus (van
Rensburg et al., 2001) and cotton (Bell et al., 2003). Such
compounds attack membranes and could be responsible
for the loss of the semi-permeability of the plasmamembrane
as found in the cell assay reported by Hamid
and Strange (2000). An attack on plasma-membranes
could also explain wilting as chickpea is likely to depend
in part of the turgor of parenchyma cells surrounding the
stele for support.CONCLUSION
There is a need to develop new control strategies
for Fusarium wilt because of the increasing importance
of Fusarium wilt in chickpea production. For instance,
the Round ReadyTM (RR) gene from Agrobacterium spp.
strain CP4 is now used widely in soybean and cotton to
prevent toxicity from the widely used herbicide
glyphosate (Bell, 2003). Similar approaches could be used
against toxins of FOC that are crucial for either
pathogenesis or increased virulence. Foreign genes could
also be introduced into biocontrol organisms to protect
them from FOC toxins and further allow destruction of
toxins in the rhizosphere. Hence, a holistic approach to
FOC toxins should facilitate development of new control
practices for Fusariumwilt of chickpea.