SUBRAMANIAM GOPALAKRISHNAN* AND VADLAMUDI SRINIVAS
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 me-tabolites provides insights into disease syndromes and may be exploited by conventional and molecular breeding to obtain crops resistant to plant disease.
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 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).
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 non-pathogenic 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 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 well-documented 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 non-hosts. 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).
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).
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).
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).
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 D max 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 plasma-membrane 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.
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.