Ascochyta blight of chickpea ( Cicer arietinum L.): a review of biology, pathogenicity, and disease management

Bassam Bayaa

Ascochyta blight (AB), caused by Ascochyta rabiei is a major disease of chickpea (Cicer arietinum L.), especially in areas where cool, cloudy, and humid weather persists during the crop season. Several epidemics of AB causing complete yield loss have been reported. The fungus mainly survives between seasons through infected seed and in infected crop debris. Despite extensive pathological and molecular studies, the nature and extent of pathogenic variability in A. rabiei have not been clearly established. Accumulation of phenols, phytoalexins (medicarpin and maackiain), and hydrolytic enzymes has been associated with host-plant resistance (HPR). Seed treatment and foliar application of fungicides are commonly recommended for AB management, but further information on biology and survival of A. rabiei is needed to devise more effective management strategies. Recent studies on inheritance of AB resistance indicate that several quantitative trait loci (QTLs) control resistance. In this p...

CSIRO PUBLISHING www.publish.csiro.au/journals/ajar Australian Journal of Agricultural Research, 2005, 56, 317–332 Ascochyta blight of chickpea (Cicer arietinum L.): a review of biology, pathogenicity, and disease management∗ S. PandeA,E , K. H. M. SiddiqueB , G. K. KishoreA , B. BayaaC , P. M. GaurA , C. L. L. GowdaA , T. W. BretagD , and J. H. CrouchA A International Crops Research Institute for the Semi-Arid Tropics, Patancheru 502 324, Andhra Pradesh, India. for Legumes in Mediterranean Agriculture, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling HWY, Crawley, WA 6009, Australia. C International Center for Agricultural Research in the Dry Areas, PO Box 5466, Aleppo, Syria. D Department of Primary Industries, Private Bag 260, Horsham, Vic. 3401, Australia. E Corresponding author. Email: s.pande@cgiar.org B Centre Abstract. Ascochyta blight (AB), caused by Ascochyta rabiei is a major disease of chickpea (Cicer arietinum L.), especially in areas where cool, cloudy, and humid weather persists during the crop season. Several epidemics of AB causing complete yield loss have been reported. The fungus mainly survives between seasons through infected seed and in infected crop debris. Despite extensive pathological and molecular studies, the nature and extent of pathogenic variability in A. rabiei have not been clearly established. Accumulation of phenols, phytoalexins (medicarpin and maackiain), and hydrolytic enzymes has been associated with host-plant resistance (HPR). Seed treatment and foliar application of fungicides are commonly recommended for AB management, but further information on biology and survival of A. rabiei is needed to devise more effective management strategies. Recent studies on inheritance of AB resistance indicate that several quantitative trait loci (QTLs) control resistance. In this paper we review the biology of A. rabiei, HPR, and management options, with an emphasis on future research priorities. Additional keywords: ascomycete, biotic stress, Didymella rabiei, epidemiology. Introduction Chickpea (Cicer arietinum L.), a self-pollinated, diploid, annual grain legume (pulse), is the third most important food legume in the world after dry bean (Phaseolus vulgaris L.) and field pea (Pisum sativum L.). It is a major source of high-quality protein in human diets and also provides high-quality crop residues for animal feed. Chickpea maintains soil fertility through biological nitrogen fixation, and contributes to the sustainability of cropping systems in cereal–legume rotations. Among temperate pulses, chickpea is the most tolerant crop to heat and drought and is suitable for production in low fertility soils. Chickpeas are of 2 types. The kabuli (garbanzo bean) types are usually large seeded, with ‘ramsowls-head’ shaped seeds, having a smooth surface and a thin cream or beige coloured seed coat. These types are grown in countries of the Mediterranean region, West Asia and North Africa (WANA), ∗ Australia, and North America. Desi types are usually small seeded, with angular seeds, reticulated (rough) seed surface, and a seed coat colour varying from yellow to black. Desi cultivars account for about 85% of the world’s total production of chickpea, and are mainly grown in the south of Asia, Iran, Ethiopia, and Mexico (Anon. 2002). In 2002, the world chickpea production was ∼7.8 million tonnes from ∼9.9 million hectares of land (FAO 2002). This constitutes ∼5% of global legume production. Average yields of chickpea vary from <400 kg/ha in Pakistan to >3600 kg/ha in China (FAO 2002). Ascochyta blight (AB), caused by Ascochyta rabiei (Pass.) Labrousse, is the most important biotic constraint for chickpea production and causes serious grain yield and quality losses (Gaur and Singh 1996b). The disease is devastating in areas where cool, cloudy, and humid weather (15–25◦ C and >150 mm rainfall) occurs during the crop season (Nene 1982) and can cause complete yield loss. This paper is one of a series of invited reviews commissioned by the journal’s Editorial Advisory Committee. © CSIRO 2005 10.1071/AR04143 0004-9409/05/040317 318 Australian Journal of Agricultural Research Geographical distribution Following the first report from north-western provinces of India (now in Pakistan), the occurrence of AB has now been reported from most chickpea-growing areas in the world (Kaiser et al. 2000a, 2000b). The disease has been reported from 34 countries across 6 continents and, as recent cultivation in Australia and Canada has shown, it can spread rapidly to new areas of chickpea production. It is the most important biotic factor affecting chickpea cultivation in areas of WANA, southern Europe (Nene 1982; Singh and Sharma 1998; Akem 1999; CAB International 2000), between 31◦ and 45◦ N, and is occasionally important between 26◦ and 30◦ N. In Australia, chickpea production increased rapidly until 1999 but was then limited by outbreaks of AB because all available commercial varieties were susceptible to the disease (Ackland et al. 1998; Knights and Siddique 2002). The disease is currently the most important yield-limiting factor, potentially affecting 95% of the chickpea area in Australia (Knights and Siddique 2002). In Western Canada, the chickpea production area increased rapidly from 800 ha in 1995 to 700 000 ha in 2000 and continued to increase, but the incidence of AB in these areas resulted in >70% yield losses (http://www.pulse.ab.ca/ascoch.pdf). The occurrence of AB has also been reported from Bulgaria (Kaiser et al. 1998) and Latin America (Kaiser et al. 2000a, 2000b). Causal organism Ascochyta rabiei, the causal agent of AB of chickpea, exists both as an anamorph and a teleomorph. The anamorph, A. rabiei, is characterised by the formation of spherical or pear-shaped black fruiting bodies called pycnidia. A pycnidium contains numerous hyaline unicellular and occasionally bicellular spores, pycnidiospores, or conidia, developed on short conidiophores (stalks) embedded in a mucilaginous mass. Pycnidiospores are oval to oblong, straight, or slightly bent at one or both ends and measure 6–12 by 4–6 µm (Punithalingam and Holliday 1972; Nene 1982). The teleomorph, Didymella rabiei (Kovacheski) var.Arx (Syn. Mycosphaerella rabiei Kovacheski), is a bipolar heterothallic ascomycete and is characterised by pseudothecia developing on chickpea crop residues that have over-wintered in the field. For successful sexual reproduction, the telomorph requires pairing of 2 compatible mating types (MAT1-1 and MAT1-2), which are widely distributed in several major chickpea-growing areas of the world (Haware 1987; Kaiser 1997; Armstrong et al. 2001). Pseudothecia are dark brown to black, subglobose, 120–270 µm in diameter, erupting from the host tissue and without a conspicuous ostiole. Binucleate asci are cylindrical to subclavate surrounded by paraphyses and contain 8 hyaline unequally bicellular ascospores. Ascospores are ellipsoid S. Pande et al. to biconic with a constriction at the septum and measure 9.5–16 by 4.5–7 µm. The fungus grows readily on a variety of nutrient media, the best being chickpea meal dextrose agar. Ascochyta rabiei generally produces a pale cream coloured mycelium in which pale brown to black pycnidia are immersed. Cultures are variable in morphology and colour, with isolates often producing a prevalence of unicellular conidia (CAB International 2000). The morphological characteristics of A. rabiei and Phoma medicaginis var. pinodella are similar, which makes it difficult to distinguish between the 2 species. However, a PCR test developed by Phan et al. (2002) can be used to detect and confirm the identity of A. rabiei. Disease symptoms Symptoms of AB can develop on all aerial parts of a plant. Seed-borne infection leads to brown lesions at the stem base of emerged seedlings. Subsequently, the lesions enlarge in size, and girdle the stem causing its breakage and death of the plant. Numerous pycnidia develop on the necrotic lesions. In the field, AB may initially appear as small patches (foci) of blighted plants, but can rapidly spread across an entire crop under favourable temperature and rainfall. Plants are attacked at any growth stage, depending on the inoculum availability. However, AB is most prominent during the flowering to early podding growth stages. Air-borne conidia and ascospores infect younger leaves and produce small water-soaked necrotic spots that rapidly enlarge and coalesce. Conidia may also be water-borne and splashdispersed to infect foliage tissue on the same or nearby plants. Subsequently, symptoms spread rapidly to all aerial parts including leaves, petioles, flowers, pods, branches, and stems, which leads to rapid collapse of tissues and death of the plant. Development of pycnidia in concentric rings on lesions is the characteristic symptom of A. rabiei infection. Lesions that develop on leaves and pods appear circular with brown margins and a grey centre that contains pycnidia, whereas lesions developing on petiole, stems, and branches are elongated. The lesions that develop on apical twigs, branches, and stems differ in size and in later stages girdle the affected plant parts. The regions above the girdled portion are killed and may break off. Diseased pods with visible blight symptoms often fail to develop any seed. Pod infection often leads to seed infection through the testa and cotyledons. Infected seed can be discoloured and possess deep, round, or irregular cankers, sometimes bearing pycnidia visible to the naked eye. Infection during the pod maturation stage often results in shrivelled and infected seed (Nene 1982; Singh and Sharma 1998; Akem 1999). Host range Artificial inoculation of A. rabiei on lentil, field pea, vetch, common bean, and cowpea revealed that the fungus is pathogenic on all these species (Zachos et al. 1963; Nene and Ascochyta blight of chickpea Australian Journal of Agricultural Research Reddy 1987; Khan et al. 1999b). A. rabiei also infects Vigna unguiculata, P. vulgaris (Kaiser 1973; Khan et al. 1999a), Lactuca serriola, Lamium amplexicaule, Medicago sativa, Melilotus alba, and Thlapsi arvense (Kaiser 1991), which are also grown in chickpea-producing regions. Pycnidial formation occurred in necrotic tissues of Medicago sativa and Melilotus alba. Ascochyta rabiei has also been isolated from Brassica nigra, Descurainia sophia, Galium apanine, Lamium amplexicaule, and Triticum aestivum, grown in fields where infected chickpea debris of the previous season remained on the soil surface during the off-season (Kaiser 1991). Pathogen variability The possible existence of different races of A. rabiei was suspected because of the variations in host–pathogen interactions and breakdown of host-plant resistance (HPR) in some cultivars at different locations. The presence of a teleomorph (D. rabiei) in the A. rabiei life cycle contributes to variability within the pathogen population, which may generate new combinations of virulence genes and thus the development of new pathotypes. However, A. rabiei is heterothallic and the 2 mating types are not present in all chickpea-growing areas (Khan et al. 1999b). Natural occurrence of the teleomorph on chickpea stubble in Australia implies that either both compatible mating types are present or that a low level of homothallic compatibility exists in A. rabiei (Galloway and MacLeod 2003). In Canada, many fields had both mating types together (Armstrong et al. 2001). 319 Pathogen variability studies based on morphological, pathogenic, and isozyme patterns, and DNA fingerprinting have been conducted in most of the major chickpea-growing countries. Based on the virulence of A. rabiei isolates on different genotypes, the existence of 2–12 races of A. rabiei has been proposed by several researchers (Table 1). In India, variations in pathogenicity among a collection of 268 A. rabiei isolates have been observed by Vir and Grewal (1974a). In Syria and Lebanon, 6 pathotypes of A. rabiei were identified using 6 chickpea differential lines (Reddy and Kabbabeh 1985). Recently, Baaya et al. (2004), using host differentials and DNA finger printing, identified a new pathotype in Syria, which can overcome the resistance of ICC 12004 and ICC 3996 chickpea lines. However, in several of these studies, no definitive relationships were observed between virulence of the isolates, their geographical origin, and morphological characteristics such as spore size, colony colour, and radial growth in vitro. Also, isozyme patterns of esterase and acid phosphatase failed to separate 15 Pakistan isolates of A. rabiei according to their aggressiveness (Hussain and Barz 1997). DNA fingerprinting has been used in an attempt to define differences among all putative races of A. rabiei. However, no definitive relationship could be observed among 48 A. rabiei isolates belonging to the 2 mating groups collected from India, Pakistan, Spain, the USA, and other countries (Navas-Cortes et al. 1998c). Randomly amplified polymorphic DNA (RAPD) analysis of A. rabiei isolates from Pakistan indicated genetic differences between isolates from the same host plant and similarities Table 1. Summary of pathogenic and molecular variability studies on Ascochyta rabiei N.B. There are few genotypes in common among the differentials used in various studies Country No. of isolates Variability parameters Italy 41 India 348 India Different isolates from Jammu region 39 Italy 30 Pathogenicity on 13 chickpea genotypes Pathogenicity on 12 differential genotypes Pathogenicity on different chickpea genotypes Pathogenicity on 11 differential genotypes Pathogenicity on differential genotypes RAPD analysis India, USA, Syria, Pakistan 47 RAPD analysis Australia, Canada, India, Syria, USA 68 RAPD analysis Australia – STMS fingerprinting USA Pakistan 102 Key findings Reference Three pathogenicity groups suggested 12 races identified Porta-Puglia et al. (1996) 10 pathotypes identified Ambardar and Singh (1996) Grouped into 11 virulent forms Grouped into 8 virulent forms No pathotype specific amplification patterns reported Isolates clustered according to the geographic origin. A DNA marker (ubc756), specific to Indian isolates identified Isolates from 4 countries clustered within major groups of Canadian isolates Diversity of Australian isolates Jan and Wiese (1991) Singh and Sharma (1998) Jamil et al. (1995) Fischer et al. (1995) Santra et al. (2000) Chongo et al. (2004) Phan et al. (2003) 320 Australian Journal of Agricultural Research between isolates from different plants and cultivars (Sarwar et al. 2000). Restriction fragment length polymorphism (RFLP) analysis of A. rabiei isolates from the Beja region of Tunisia indicated a low correlation between their virulence and RFLP patterns (Hamza et al. 2000). Phan et al. (2003), using the sequence tagged microsatellite site (STMS) fingerprinting technique, attempted to study A. rabiei diversity and its populations in Australia. All these studies identified specific DNA fragments that may be used as isolate-specific genetic markers in sexual crosses. In an analysis of micro- and macro-geographical variations of A. rabiei using DNA fingerprinting, Morjane et al. (1997) observed that 17 different fungal genotypes were distributed at different frequencies in the 5 fields sampled, of which 2 were common in all the fields. In a few instances, more than one fungal genotype was isolated from the same plant. Higher levels of pathogen diversity were found within rather than between locations, and different genotypes from a particular location were not obviously related to each other. A combination of RAPD analysis and RFLP analysis using an oligonucleotide probe complementary to the microsatellite sequence (GATA)4 distinguished variability within and among the major pathotypes of A. rabiei. A combination of microsatellites and RAPD markers distinguished the earlier identified 4 pathotypes of A. rabiei (Weising et al. 1991) into 5 pathotypes, which were further resolved into several genotypes, indicating that different isolates of a pathotype do not have clonal lineages (Udupa et al. 1998). In all the above-mentioned studies, the DNA amplification patterns of A. rabiei isolates were not correlated with their grouping into different pathogenic groups. A standard set of international differential lines, which clearly distinguish all A. rabiei isolates from a broad geographical area, may help in the identification of different races of A. rabiei, if they do indeed exist. Epidemiology of the disease Pathogen survival Ascochyta rabiei survives either on or in seed or plant debris in the form of mycelium, pycnidia, and various teleomorphic stages (Kaiser 1997). Didymella rabiei can survive in a free state in the soil. The teleomorph was first discovered on over-wintered infested chickpea debris from a field near Genesee, Idaho, USA, in 1986 (Kaiser 1994). At temperatures of 10–35◦ C, A. rabiei can survive for 8 months in infected chickpea debris (Nene and Reddy 1987), 20 months on infected stem (Kaiser and Hannan 1987) and 5 months on the surface of chickpea seed (Singh et al. 1995). However, when infected debris and stems were buried in soil, the pathogen survival was drastically reduced. When the infected seeds were S. Pande et al. stored at 4◦ C, A. rabiei remained infective for 13 years (Kaiser 1997). The teleomorph helps in long-term survival of the pathogen, but this stage has never been observed on newly infected plants. However, in many regions, pseudothecia can often be found on infected crop debris. Low temperature and high moisture are essential for initiation and development of pseudothecia (Trapero-Casas and Kaiser 1992b; Navas-Cortes et al. 1998a). The density of asci and ascospore production per pseudothecium, and conidia per pycnidium were much higher in cool climatic conditions than in warmer conditions (Navas-Cortes et al. 1998b). Ascospores are also important in long-distance dispersal of the pathogen (Trapero-Casas et al. 1996). Relative humidity rather than temperature was the critical factor determining the development of psuedothecia and pycnidia on crop debris. Thus, at lower humidities (such as 86% RH) the development of A. rabiei on debris was very limited irrespective of temperature (Navas-Cortes et al. 1998a). When the debris was buried in soil, pycnidia rather than pseudothecia were produced, and the developed pseudothecia were degenerated and contained a reduced number of asci (Navas-Cortes et al. 1995). Perpetuation of A. rabiei through crop debris in tropical countries may be influenced by the high temperature and low rainfall during out-of-season summer months, which decrease the survival of A. rabiei in crop debris. It is notable that the effect of light on in vitro pseudothecial development was negligible and had little effect on the pattern and quantity of ascospores discharged. Disease spread Seed transmission of A. rabiei and air-borne spores can lead to disease spread and establishment of compatible mating types in new areas and thus the development of the teleomorph. Seed transmission ensures random distribution of the pathogen in a field, providing many primary infection foci. Movement of infected chickpea seed is responsible for introducing AB into Canada, Iran, Australia, and the USA (Kaiser 1997). Maden et al. (1975) detected A. rabiei in 70% of the chickpea seed samples from central Anatalia-Turkey, with seed infection ranging from 1–16%. Conidia and ascospores are responsible for secondary spread of the disease. Subsequent wetting, rain splash, and strong winds disperse conidia developed on diseased plant parts, particularly if conidia are contained in droplets. Ascospore production on highly infected crop residues reaches up to 1.5 × 104 ascospores/mm2 of tissue surface. Under moist conditions, the asci protrude through the opening of a mature pseudothecium and forcibly discharge ascospores into the air (Trapero-Casas and Kaiser 1992b). At 15–25◦ C, >70% of ascospores were discharged from mature pseudothecia within 2 h of wetting and served as primary inoculum. Ascochyta blight of chickpea Frequency of infection cycles occurring during a growing season is influenced by environmental conditions and cultivar susceptibility (Nene and Reddy 1987). Disease development Ascochyta blight infection and disease development occur at a temperature range of 5–30◦ C with an optimum of 20◦ C, and 17 h of wetness is essential to produce severe infection. Dry periods (6–48 h) immediately after inoculation sometimes increase disease severity; however, dry periods of >12 h after an initial wetting period of 6 h usually have an adverse effect on disease development (Trapero-Casas and Kaiser 1992a). Jhorar et al. (1998) observed that dry periods immediately after inoculation followed by a wetness period reduced disease severity, and reduction in disease severity was correlated with an increase in the dry period. Little infection developed without leaf wetness even when the RH was 98% and no infection developed when the RH was <95%. Disease severity increases with increasing periods of darkness after inoculation. When leaf wetness was maintained over an 8-day period, there was an increase in the number of pycnidia and production of conidia on infected leaves (Jhorar et al. 1998). Under cool weather, spread and development of AB in a maturing crop can be rapid, with an incubation period as short as 6 days (Pandey et al. 1987). Plant age was observed to have a profound effect on susceptibility of different chickpea genotypes to AB infection, with plants at podding stage being most susceptible (Chongo and Gossen 2001). Disease prediction models Disease prediction models based on the climatic factors that favour development of AB have been used to assess the disease risk for various agrogeographical zones and different growth stages. Comparisons between AB incidence and weather variables over a 15-year period, at 2 different locations (one with a regular disease incidence and the other with no reports of disease incidence), showed that maximum temperature and afternoon RH were the 2 most important variables for disease prediction. A ratio of these two weather variables, referred to as humid thermal ratio (HTR), was the best predictor of outbreaks of AB (Jhorar et al. 1997). Although several studies have been conducted to determine epidemiological factors that favour AB development, many gaps still exist in our understanding of disease development and prediction of epidemics. Hence, more systematic evaluations of host-pathogen interactions, disease perpetuation and dissemination, sources of inoculum, host range, and favourable environmental (weather) conditions are needed to fill the knowledge gaps in disease prediction. Australian Journal of Agricultural Research 321 Pathogenicity The infection process of A. rabiei on leaves and stems of both resistant and susceptible genotypes has been well studied. Production of toxins, cell wall degrading enzymes, and degradation of host phytoalexins are responsible for pathogenicity of A. rabiei. Histopathology Germination of A. rabiei spores occurs at 12–48 h after inoculation (HAI). Germ tubes further elongate and form ramifications on the leaf surface. Hyphal branches form appressoria-like structures at their tips, which are separated from the germ tube by a septum and the hyphae covered by a mucilaginous exudate (Hohl et al. 1990). A. rabiei penetrates directly by mechanical force through the cuticle between 2 epidermal cells. For a short distance, hyphae push forwards subcuticularly along the junction of epidermal cells before proceeding inward (Pandey et al. 1987; Hohl et al. 1990). Near a stoma, hyphae penetrate through a juncture of guard and subsidiary cells, even when a stoma is open (Pandey et al. 1987). By using Gus (β-glucuronidase)-transformed A. rabiei, which did not differ significantly from the parent strain in production of hydrolytic enzymes and toxins, it was confirmed that fungal penetration occurs directly through the cuticle. Penetration through hydathodes has also been observed (Kohler et al. 1995). After penetration, hyphae grow parallel between epidermal and palisade parenchyma cells, disintegrating the inner structure of leaves. Hyphal diameter measures up to 2 µm outside the leaf, and up to 3.5 µm inside the leaf. Subsequently, subepidermal mycelia form dark aggregates. Epidermal cells collapse, and cells of palisade and spongy parenchyma lose their shape and organisation. The entire cortex and part of the pith disintegrate completely by the fifth day after inoculation (DAI). Hyphae aggregate and form pycnidia that emerge by collapse of the surrounding leaf tissues. From pycnidium, conidia ooze out through an ostiole (Pandey et al. 1987; Hohl et al. 1990). By the seventh DAI, most of the non-lignified tissues are destroyed and necrosis is much more evident. Lignified tissues, particularly xylem tracheary elements, remain unaffected (Pandey et al. 1987). Fungal growth proceeds from the leaflets to stems through petioles. Within the petioles, A. rabiei rarely colonises xylem vessels but colonises phloem vessels and the petioles break off (Kohler et al. 1995). The fungus invades and colonises xylem and phloem vessels of the stem, but walls of these vessels remain intact. Pycnidia form subepidermally within cortex and pith. In resistant genotypes, a strong autofluorescence typical of a hypersensitive response (HR) was observed in leaf and stem tissues in early stages of infection. As a result, no hyphae could be observed in girdled stems (Hohl et al. 1990). 322 Australian Journal of Agricultural Research Pathogen toxins and enzymes involved in infection Extensive disintegration of parenchymatous cortical and pith tissues that occurs in advance of invading fungal hyphae during the infection process indicates involvement of toxins and cell wall-degrading enzymes produced by A. rabiei in its pathogenesis (Pandey et al. 1987; Hohl et al. 1990). The toxins solanopyrone A, B, and C are involved in pathogenicity of A. rabiei. Application of purified solanopyrones to chickpea leaves produced visible symptoms in 24 h, followed by contraction of protoplasts of epidermal, palisade, and spongy parenchyma cells (Hohl et al. 1991). There was a good correlation between the in vitro production of solanopyrones by different isolates of A. rabiei and their pathogenicity (Kaur 1995). Ascochyta rabiei, when grown on plant sap medium, produced an additional heat-labile toxic polypeptide of 14 amino acids consisting of a glycosidic moiety with a molecular weight of 7.6 k Da. Production of this polypeptide peaked at 4 days, with 166.7 units/mL culture filtrate (Chen and Strange 1994). Ascochyta rabiei degrade phytoalexins produced in chickpea plants by converting the pterocarpans into 2′ -OH isoflavans and 1a-OH pterocarpdiens. These two enzymes required for this conversion, a reductase and a hydroxylase are expressed constitutively in A. rabiei (Tenhaken et al. 1991). These two enzymes are specific for (–) isomers of phytoalexins such as maackiain and medicarpin (Weltring et al. 1995). Other pathogenic enzymes such as cutinase (Tenhaken et al. 1997) and a polygalacturonase that degrades the polygalacturonic acid but not pectin were purified from the culture filtrate of A. rabiei. Purified polygalacturonase released no oligo-galacturonides that elicit chickpea plants and trigger a defence response (Tenhaken and Barz 1991). Host plant resistance Ascochyta blight resistance of chickpea is determined by a diverse set of anatomical, biochemical, physiological, and genetic characters. Host metabolitic activities that inhibit the pathogen invasion include induction of hypersensitive response (HR), cell wall reinforcement by deposition of callose, lignin, esterbound cinnamic acids/polyphenols, and hydroxyproline-rich glycoproteins, induction of phytoalexins, and proteins that inhibit the pathogen growth or reduce its virulence. Anatomical characters Anatomical characters such as thickness of the leaf cuticle, stem cuticle, epithelium, and palisade cells provide a mechanical barrier for pathogen penetration. A higher number of xylem elements and xylem parenchyma cells, and thicker stem epidermis and hypodermis exist in resistant S. Pande et al. genotypes than in susceptible genotypes (Angelini et al. 1993). Susceptible genotypes had a thinner outer cell wall and smaller area of cell lumen in the second outer cell layer (Venora and Porta-Puglia 1993). The cortical region was thinnest in the susceptible genotype Aug 424, and A. rabiei caused greater damage to cortical and pith tissues by the third DAI (Sarwar et al. 1996). Host physiology Ascochyta blight resistance in chickpea genotypes correlates positively with their respiration rate and total carbohydrate content. In a resistant genotype the rate of respiration and total carbohydrate content increased by the second DAI due to a HR, whereas in a susceptible genotype this increase occurred only by the fifth DAI (Dolar and Gurcan 1995). Total and reducing sugars, phosphorous, and potash gradients increased more in resistant than in susceptible genotypes (Khirbat and Jalali 1999). Hypersensitive response An incompatible plant–pathogen interaction quite often results in a rapid HR. When infected with A. rabiei, a rapid HR-like browning reaction developed in the leaves of resistant genotype ILC 3279 by the second DAI, whereas such changes were not observed in the susceptible genotype ILC 1929 (Hohl et al. 1990). Similar responses were observed when crude culture filtrate (CCF) of A. rabiei was applied to cell cultures of these 2 genotypes. In cells of ILC 3279, browning became visible 5–7 h after application of CCF. The browned cells did not develop a red stain after treatment with phloroglucinol-HCl, indicating the absence of lignin-specific compounds. Occurrence of HR was further confirmed by determining cell death by fluorescein diacetate staining. Cells of ILC 3279 died rapidly at 12 HAI but there was no cell death in ILC 1929. Lack of HR in cells treated with autoclaved or proteinase K-treated CCF suggested the proteinaceous nature of the HR inducer (Vogelsang et al. 1994). Phytoalexin accumulation Phytoalexins are metabolic compounds that have an important role in the defence mechanisms of higher plants towards phytopathogenic fungi. In chickpea genotypes resistant to A. rabiei infection, pterocarpan phytoalexins, (–) medicarpin and (–) maackiain, were produced rapidly and in higher quantities than in susceptible genotypes in response to attack by A. rabiei. Detection of maackiain alone in the resistant genotype indicates its important role in disease resistance (Dolar and Gurcan 1993). Treatment of cell cultures of ILC 3279 with CCF of A. rabiei resulted in accumulation of medicarpin as a major ester-bound component in the cell wall 12 h after treatment, whereas there was no accumulation of medicarpin in ILC 1929 cells (Vogelsang et al. 1994). Ascochyta blight of chickpea Phenolic compounds Main constitutive phenolic compounds in chickpea are biochanin A (5,7-dihydroxy-4′ -methoxyisoflavone) and formononetin (7-hydroxy-4′ -methoxyisoflavone). These isoflavones occur as aglycones (7-O-glucosides), most prominently as 7-O-glucoside-6′ -O-malonate esters (Koster et al. 1983). When challenged with A. rabiei or its CCF, resistant genotypes rapidly accumulated large quantities of phenolic compounds compared with susceptible genotypes (Vir and Grewal 1974b; Sindhu et al. 1995; Khirbat and Jalali 1997). Also, an elicitor preparation from CCF of A. rabiei, enhanced levels of formononetin and biochanin A in sliced cotyledons of chickpea (Kessmann and Barz 1986). Defence-related enzymes Induction of fungal cell wall-degrading hydrolytic enzymes, and enzymes of the phenylpropanoid pathway and cell wall lignification have a role in conferring AB resistance to chickpea. With the addition of CCF of A. rabiei, activity of phenylalanine ammonia lyase (PAL) increased by about 20-fold in cell culture of chickpea line ILC 3279, but there was no change in PAL activity of ILC 1929 cell culture (Vogelsang et al. 1994). Following A. rabiei inoculation, the activity of chitinase in leaves of the resistant genotype (E 100 Y) increased 5-fold by the sixth DAI, compared with the uninoculated control. Further, induction of chitinase was also higher in excised infected pods of resistant genotypes compared with susceptible genotypes (Nehra et al. 1994). Peroxidase is essential for lignosuberisation, which occurs in cell walls in response to pathogen invasion. Diamine oxidase involved in polyamine catabolism is the main source of H2 O2 , which is essential for peroxidase activity. Activities of these 2 enzymes in chickpea stems increased during A. rabiei infection, and increase was greater in resistant compared with susceptible genotypes. In lignosuberised barriers set up by cortical and pith parenchyma cells in response to pathogen invasion, apparent histochemical activities of both these enzymes were detected (Angelini et al. 1993). Accumulation of peroxidase and β-1,3-glucanase, a fungal cell wall hydrolytic enzyme, was higher in resistant genotypes than in susceptible ones, when grown in the presence of CCF of A. rabiei (Sindhu et al. 1995). Disease management Identification of host plant resistance The preliminary step for exploiting HPR is the development of reliable and repeatable techniques for large-scale screening of germplasm and breeding lines. Several techniques suitable for AB resistance screening under field and greenhouse conditions have been developed (Nene 1982; Weising et al. 1991; Nasir et al. 2000; Bretag and Meredith 2002; Australian Journal of Agricultural Research 323 Bretag et al. 2002a, 2002b). Resistance screening using cut-twig and detached-leaf techniques correlated with greenhouse screening. These quick and reliable methods are useful in screening segregating lines derived from wide hybridisation, since whole plants can then be used to screen for other target traits including seed production (Sharma et al. 1995). Field screening Field screening of chickpea genotypes for AB resistance as standardised by ICRISAT and ICARDA involved planting the test material with a 40-cm row space and interplanting a susceptible cultivar (e.g. L 550 or Pb 7 or ILC 1929), which serves as an indicator/spreader line, after every 4–8 rows. Infected debris is scattered between rows, and at flowering the plants are inoculated with a spore suspension (∼105 spores/mL) in the evening on cloudy days. Following inoculation, sprinkler irrigation for 15 days is provided during dry weather. The disease rating scale commonly followed is a 1–9 scale, where 1 is no visible lesions on any plants and 9 is profuse lesions on all plants, stem girdling on more than 50% of the plants, and many plants killed (Singh et al. 1981; Reddy and Singh 1984). Controlled environment screening Different screening techniques have been developed at various research centres, for artificial resistance screening of chickpea germplasm against A. rabiei. A controlledenvironment facility established at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, India, facilitates reliable large-scale screening for AB resistance. Ten-day-old chickpea seedlings grown in plastic trays (35 × 25 × 8 cm) in a mixture of sterilised river sand and vermiculite (3 : 1), were transferred to a plant growth chamber in which air temperature was maintained at 20 ± 1◦ C. The seedlings were inoculated by spraying a conidial suspension (5 × 104 conidia/mL) of A. rabiei. The conidia were produced on chickpea seed and harvested into sterile distilled water. In the growth chamber, leaf wetness was maintained up to 72 HAI and RH maintained at 65–70% during the subsequent 12 days. A 12-h photoperiod was provided with fluorescent lights. Disease severity was scored on a 1–9 rating scale at 14 DAI (Haware et al. 1995). Recently, an improved 0–9 rating scale for controlledenvironment screening of AB infection has been developed by Chongo et al. (2004). Cut twig method Long, tender shoots cut from the test plants are wrapped with a cotton plug and transferred to a test tube (15 × 100 mm) containing fresh tap water. Twigs are inoculated by spraying conidial suspension (4 × 104 conidia/mL) of A. rabiei and kept in moist 324 Australian Journal of Agricultural Research S. Pande et al. chambers for 72 h. After 72 h of leaf wetness with >90% RH, infected plants are incubated for another 13 days and then assessed for disease. The results obtained by this method were well correlated with those of greenhouse and field-screening techniques (Sharma et al. 1995). Detached leaf/leaflet technique Surface-sterilised whole leaves are transferred onto water agar in 90-mm Petri dishes and inoculated by spraying the dishes with a spore suspension. The lids of the Petri plates are sealed with paraffin wax and incubated at 20◦ C with a 12-h photoperiod. Inoculated leaves are observed for disease development on the eighth DAI (Singh and Sharma 1998). Leaflets from the most recent fully expanded leaves are collected from 15-day-old chickpea plants. The detached leaflets are floated, lower surface down, on tap water inside 90-mm Petri dishes, and upper surfaces of the leaflets inoculated with 5 µL of A. rabiei. The leaflets are incubated for 14 days at 20 ± 2◦ C, with a 12-h photoperiod. Disease severity scores are based on the number of leaflets infected and lesion size (Dolar et al. 1994). The disease ratings obtained from this technique are in correlation with whole-pot screening methods. Resistant sources Deployment of resistant genotypes is the most effective way to minimise yield losses due to AB. In several studies conducted in different chickpea-growing areas of the world, several sources of resistance to AB were identified (Table 2). Few of the resistant sources were also released as cultivars (Table 3). Furthermore, development of AB-resistant genotypes has made it possible to sow the crop during winter in the Mediterranean region thereby doubling the chickpea production potential. High levels of AB resistance have been identified among annual wild Cicer spp., accessions of C. bijugum, C. judaicum, and C. pinnatifidum (Singh et al. 1981; Singh and Reddy 1991; Collard et al. 2001) and there is potential to transfer resistance genes from these species into C. arietinum. One hundred and twenty-eight wild accessions of chickpea belonging to 8 species were screened for AB resistance under controlledenvironment conditions at ICRISAT, Patancheru, India. One accession each of C. bijugum and C. pinnatifidum, 2 of C. cuneatum and 17 of C. judaicum were resistant to AB infection, with a mean disease score of ≤3.0 on a 1–9 rating scale. Another 18 accessions of C. judaicum and 8 accessions of C. pinnatifidum were moderately resistant to AB infection, with a mean disease score of 3.1–5 (Table 4). At the International Center for Agricultural Research in the Dry Areas (ICARDA), Syria, >25 000 chickpea lines have been screened for AB resistance and 14 durable sources of resistance have been identified. ILC 200, ICC 4475, ICC 6328, ILC 6482, and ICC 12004 were found to be resistant to 6 races of A. rabiei in repeated field and greenhouse evaluations (Singh and Reddy 1993). Several of the resistant sources identified and breeding lines derived from them have been released worldwide (Table 3). In total, 1584 AB-resistant chickpea Table 2. Sources of resistance to Ascochyta blight in chickpea germplasm Genotype ICC 3634, ICC 4200, ICC 4248, ICC 5124, ICC 6981, ILC 196, ILC 3346, ILC 3956, ILC 4421 ILC 72, ILC 191, ILC 3279, ILC 3856 ICC 76, ICC 187, ICC 607, ICC 1121, ICC 1136, ICC 1416, ICC 1754, ICC 1762, ICC 1903, ICC 7773, ILC 236, ILC 482, ILC 484, ILC 2548, ILC 2956 ICC 4000, 4014 ILC 3864, ILC 3870, ILC 4421 ILC 190, ILC 201, ILC 202, ILC 2506, ILC 3856, ILC 5928, ICC 3996, FLIP 83-48 ILC 5586, ILC 5894, ILC 5926, ILC 6482, ILC 7795, ICC 4475, ICC 6328, ICC 12004 ILC 3287 CG 715, ACC 76, H 86-8, H 86-100, HK 86-120 ILC 3896, ICC 7514, NEC 123, P 1279-2, P 4268-1 ICC 8161 ICC 1278, ICC 1284, ICC 1285, ICC 1304 FLIP 92-262C, FLIP 92-110C, FLIP 92-154C Remarks Reference Reddy and Singh (1984) Resistant in 8 chickpea-growing countries (including India, Pakistan, and Mediterranean region) ILC 482 and ICC 1903 were always rated 1 on a 1–9 scale Singh et al. (1984) Both foliage and pods of ICC 4000 were resistant to Ascochyta blight Singh and Kapoor (1985) Katiyar and Sood (1985) Resistant to 3–6 races of A. rabiei Pal and Singh (1990) Singh and Reddy (1990) Resistant both in greenhouse and field Singh and Reddy (1992) Rate-reducing phenomenon of Ascochyta blight observed Reddy and Singh (1993) Singh and Pal (1993) Gaur and Singh (1996a) Shukla and Pandya (1988) Wadud and Riaz (1988) Toker et al. (1999) Ascochyta blight of chickpea Australian Journal of Agricultural Research Table 3. Some chickpea lines released in different countries, with acceptable level of resistance to Ascochyta blight Source: Anon. (2002) Accession Country of origin Country of release Released name Year of release ILC 72 ILC 72 ILC 195 ILC 195 ILC 195 ILC 200 ILC 202 ILC 237 ILC 411 ILC 464 ILC 482 ILC 482 ILC 482 ILC 482 ILC 482 ILC 482 ILC 482 ILC 482 ILC 482 ILC 484 ILC 533 ILC 915 ILC 1335 ILC 2548 ILC 2555 ILC 3279 ILC 3279 ILC 3279 ILC 3279 ILC 3279 ILC 3279 ILC 3279 ILC 3279 ILC 3279 ILC 6188 n.a. n.a. USSR USSR USSR USSR USSR Spain Iran Turkey Turkey Turkey Turkey Turkey Turkey Turkey Turkey Turkey Turkey Turkey Egypt Iran Afghanistan USSR Ethiopia USSR USSR USSR USSR USSR USSR USSR USSR USSR France Italy Spain Egypt Morocco Turkey Spain China Oman China Cyprus Algeria France Iran Iraq Jordan Lebanon Morocco Syria Turkey Libya Georgia Sudan Sudan Spain Spain Algeria China Cyprus Iran Iraq Italy Jordan Syria Tunisia Italy Califfo Fardan Giza 195 ILC 195 ILC 195 Zegri ILC 202 ILC 237 ILC 411 Kyrenia ILC 482 TS 1009 ILC 482 Rafidain Jubeiha 2 Janta 2 ILC 482 Ghab 1 Guney Sarisi 482 ILC 482 Elixir Jebel Marra-1 Shendi Almena Alcazaba ILC 3279 ILC 3279 Yialosa ILC 3279 Dijla Sultano Jubeiha 3 Ghab 2 Chetoui Ali 1990 1985 1995 1986 1986 1985 1988 1988 1988 1987 1988 1988 1995 1992 1990 1989 1986 1986 1986 1993 2000 1994 1987 1985 1985 1988 1988 1984 1995 1992 1990 1990 1986 1987 1998 n.a., Not available. lines were developed with a range of maturity, plant height, and seed size not previously available to growers in the blight-endemic areas in the Mediterranean region. These included 92 lines resistant to 6 races of A. rabiei (Singh and Reddy 1996). Breeding for disease resistance Conventional breeding Ascochyta blight resistance breeding commenced in India in the early 1930s and the first resistant cultivar was developed and released about 60 years ago (Luthra et al. 1941). Later reports from the Soviet Union (Gushkin 1946) announced the development and release of 3 cultivars, viz. Skorospelka, Alpha, and Mogucii, resistant to AB. In contrast, no AB-resistant cultivars were released in the Mediterranean region until 1984. There has been slow 325 Table 4. Screening wild Cicer spp. for Ascochyta blight resistance at ICRISAT, Patancheru, India Rating scale for Ascochyta blight on chickpea seedlings (modified from Jan and Wiese 1991): 1, no symptoms; 2, minute lesions prominent on the apical stem; 3, lesions up to 5 mm size and slight drooping of the apical stem; 4, lesions obvious on all plant parts, and clear drooping of apical stem; 5, lesions obvious on all plants/parts, defoliation initiated and breaking and drying of branches slight to moderate; 6, lesions as in 5, defoliation, broken, dry branches common, some plants killed; 7, lesions as in 5, defoliation, broken, dry branches very common, up to 25% of the plants killed; 8, symptoms as in 7 but up to 50% of the plants killed; 9, symptoms as in 7 but up to 100% of the plants killed. Based on the disease score, the wild accessions were categorized for their reaction to Ascochyta blight infection as follows: 1, immune (I); 1.1–3, resistant (R); 3.1–5, moderately resistant (MR); 5.1–7, susceptible (S); and 7.1–9, highly susceptible (HS) Cicer species C. arietinum C. bijugum C. cuneatum C. echinospermum C. judaicum C. pinnatifidum C. reticulatum C. yamashitae Total No. of lines screened 3 18 2 2 46 26 26 5 128 Reaction to Ascochyta blight infection I – – – – – – – – – R – 1 2 – 17 1 – – 21 MR – – – – 18 8 – – 26 S – 4 – 2 11 7 16 1 41 HS 3 13 – – – 10 10 4 40 progress due to the lack of a simple resistance screening technique, unavailability of germplasm sources with a high level of resistance, and the evolution of new races of A. rabiei. At ICARDA, hybridisation work, which was initiated in 1978, attempted to combine high yield with resistance to cold and AB. Using off-season advancement facilities at Terbol in Beqa’a valley in Lebanon, more than 3000 AB-resistant and high-yielding lines have been bred between 1981 and 2002 and freely shared (Malhotra et al. 2003). After the initial success, most of the previously released cultivars have succumbed to new races/pathotypes of A. rabiei, resulting in short life span for resistant cultivars (Malhotra et al. 2003). The bulk-pedigree method to breed AB-resistant chickpeas was in vogue at ICARDA until 1998 when studies revealed that the efficiency of selection for AB resistance and large seed size was improved with single seed descent (SSD) at F2 and F3 and pedigree method from F4 (R. S. Malhotra, ICARDA, pers. comm.). This combination of SSD and pedigree method has resulted in good progress in AB resistance breeding. Attempts have been made to combine genes that may confer resistance against several races of A. rabiei in one line. Chickpea breeders at ICARDA have been successful in pyramiding a few genes from different sources using a stepwise breeding program. A good number of improved 326 Australian Journal of Agricultural Research lines, which may posses at least 4 or 5 genes for AB resistance from different genetic backgrounds are now in the final stages of development prior to being tested on a large-scale (Malhotra et al. 2003). ICRISAT has concentrated on development of ABresistant lines in desi chickpea. Multiple crosses have been used to accumulate resistance genes from diverse sources. Many of the advanced breeding lines developed from this program have shown resistance to all 4 isolates of A. rabiei tested under controlled-environment screening (ICRISAT 2003). Resistance to AB has been one of the major objectives in chickpea breeding programs of many countries, such as Canada, the USA, Australia, Turkey, and Pakistan. Germplasm and breeding lines supplied by ICARDA and ICRISAT have been widely used as sources of AB resistance. In the USA, development of AB-tolerant kabuli chickpea varieties such as ‘Dwelley and Sanford’ in early 1990s, helped in reducing damage from AB devastation. In recent years, 2 additional varieties, Evans and Sierra, with good levels of resistance to AB, have been released (http://pwa.ars.usda.gov/pullman/glgp/variety.html). In desi chickpea, an ICRISAT-derived line, ICCV 92809, with early maturity and good level of resistance to AB was released with the name ‘Myles’. This variety was also well adapted to western Canada and spread rapidly there. The crop development centre (CDC), Saskatoon, has developed 4 AB-tolerant cultivars in desi type (CDC Anna, CDC Cabri, CDC Desiray, and CDC Nika) and 1 cultivar in kabuli type (CDC Frontier) (Warkentin et al. 2004). In Australia, the first variety with moderate resistance to AB was the desi type cultivar ‘Howzat’ released in 2001. Australian chickpea breeders have further selected a number of desi and kabuli lines having moderate to high levels of AB resistance. These include breeding lines from ICRISAT (e.g. ICCV 96836) and ICARDA (e.g. FLIP94-508C, FLIP94-90C, FLIP 94-92C, S95362, and S95342) and selections from existing Australian varieties (e.g. Heera, Sona, and Barwon) and breeding lines (Materne et al. 2002). Some of these lines are in their final stages of testing and will soon be commercialised to help revive the local chickpea industry. The area cropped to chickpea in Australia is expected to increase to at least 500 000 ha once AB-resistant cultivars become widely available. Mutation breeding has been successfully used to develop AB-tolerant varieties in Pakistan. The first variety, CM 72 (desi type), from this program was developed in 1983 and helped the chickpea industry to survive. The other mutant varieties later released included CM 88 and CM 98 in desi type and CM 2000 in kabuli type (http://www.niab.org.pk/mutation.htm). Several other AB-tolerant varieties, such as Dashat S. Pande et al. and NIFA 88, have been developed through conventional breeding methods. In the absence of highly resistant sources, no single strategy in breeding for AB-resistant cultivars is likely to succeed. A combination of different strategies needs to be developed and utilised. The release of several cultivars, possibly with known reactions in different races/pathotypes, will be useful in case the resistance breaks down in one of the cultivars. Marker-assisted breeding Molecular markers linked to major quantitative trait loci (QTLs) contributing resistance have been discovered and may be used in marker-assisted breeding for resistance to AB. The markers will be important in enabling the pyramiding of resistant genes from diverse sources and should significantly reduce the time required in the development of resistant cultivars. Deoxyribonucleic acid markers will also encourage the use of exotic sources of disease resistance by dramatically improving the pace and precision of recovering the recurrent parent genome in backcross programs. Most importantly, DNA markers may help break deleterious linkage drag associated with introgressing resistance genes from wild species. Considerable progress has been made in mapping of QTLs conferring AB resistance in chickpea. Using a recombinant inbred line (RIL) population from an interspecific cross of C. arietinum (FLIP84-92C, resistant parent) × C. reticulatum (PI 599072, susceptible parent), Santra et al. (2000) identified 2 major QTLs (QTL 1 and QTL 2), which accounted for >45.0% of the estimated phenotypic variation for AB resistance, and mapped these QTLs to linkage groups 6 and 1, respectively. Two RAPD markers flanked QTL 1 and were 10.9 cM apart, whereas 1 inter simple sequence repeat (ISSR) marker and 1 isozyme marker flanked QTL 2 and were 5.9 cM apart. From the same mapping population, Tekeoglu et al. (2002) reported that QTL 1 is linked to the microsatellite and ISSR markers GAA 47, ubc 733 and ubc 181, whereas QTL 2 is linked to microsatellite markers Ta 72A, Ta2, Ts 54, and Ta 146. Genetic basis of host–pathogen interaction Detailed information on the number, nature, and diversity of genes conferring resistance is a prerequisite for exploiting a particular genotype in resistance breeding programs. Initial studies suggested that AB resistance of chickpea is due to either a single dominant or a recessive gene (Singh and Reddy 1991). Depending on the mode of inheritance of resistance to AB in F1 and F2 generations, Singh and Reddy (1983) concluded that the resistance in ILC 72, ILC 183, ILC 200, and ILC 4935 was due to a single dominant gene, and in ILC 191 to a single recessive gene. Allelic studies by Tewari and Pandey (1986) indicated the presence of 3 independently segregating dominant genes for resistance Ascochyta blight of chickpea in P 1215-1, EC 26446, and PG 82-1, and a recessive gene in BRG 8. However, 2 dominant complementary genes were reported to control disease resistance: Arc1 and Arc2 in genotype GLG 84038, and Arc3 and Arc4 in GL 84099. Similarly, the resistance in ICC 1468 has been reported to be controlled by 1 dominant gene (Arc5(3,4) ) and 1 recessive gene (Arc1 ). In these 3 genotypes, inter-allelic interactions, additive gene effects, and dominance influenced the resistance (Dey and Singh 1993). Recent studies on RILs suggest that several QTLs are involved in controlling resistance to AB. Three sets of RILs derived from 2 intraspecific crosses, PI 359075(1) × FLIP 84-92C(2) and Blanco Lechoso × Dwelley, and 1 interspecific cross, FLIP 84-92C(3) × C. reticulatum (PI 489777), were developed at ARS-USDA, Pullman, WA (http://www.nps.ars.usda.gov/). Evaluation of disease response in these RILs indicated that 3 recessive complementary major genes with some modifiers conferred AB resistance. Absence of 1 or 2 of the major genes confers susceptibility while presence of the modifiers determines the degree of resistance (Tekeoglu et al. 2000). Conversely, 6 QTLs for AB resistance were identified in 3 regions of the genome of an intraspecific population. The major QTLs generally showed additive gene action, as well as dominance inter-locus interaction in the multiple genetic model (Flandez-Galvez et al. 2003a). Other studies report 2–6 major QTLs with various different effects and interactions (reviewed by Millan et al. 2005). These different estimates of the genetic basis of AB resistance result from the use of different fungal isolates and host genotypes. Clearly, AB resistance breeding is a complex endeavour, as any new cultivar needs to carry resistance genes effective against a range of AB isolates. However, these studies seem to suggest that there is a range of different sources of resistance. Pyramiding of different resistance genes may facilitate building up the level of resistance and increasing the durability of that resistance. Studies conducted in Australia (Collard et al. 2003; Flandez-Galvez et al. 2003a, 2003b) also indicated involvement of QTLs for AB resistance. Two sets of mapping populations were used: RILs developed from an intraspecific cross involving a highly susceptible cultivar Lasseter and a resistant line ICC 12004, and an F2 mapping population derived from a cross between the susceptible cultivar Lasseter and a resistant C. echinospermum accession PI 527930. Seven QTLs were identified for AB resistance and mapped on the linkage map. Two QTLs were associated with resistance at the seedling stage and 2 others were associated with adult plant resistance. Resistance Gene Analogue (RGA) and STMS markers closely flanking major resistance QTLs were identified. Two markers (CLRRinv and TA146) flanked the strongest QTL (QTL 3) at an interval of 0.1 cM. QTL 5 and QTL 7 were flanked by STMS markers, Australian Journal of Agricultural Research 327 which were 1.9 (TS 12, TR 56) and 7.6 cM (M44 sp, TA 28) apart, respectively. After validation, these flanking markers may be used in marker-assisted selection to breed for elite chickpea cultivars with durable resistance to AB. The tight linkage of RGA markers to the major QTLs will also allow map-based cloning of the AB resistance genes. Cultural control Cultural practices that reduce the main sources of inoculum are most important in effective disease management. Planting healthy seed, crop rotation with non-host crops such as cereals, destruction of chickpea stubble, and deep sowing are all important measures to reduce the amount of inoculum and the likelihood of an AB epidemic. Under low disease pressure, agronomic practices such as delayed sowing, lower seed rate, and wider row and plant spacing can reduce the incidence and severity of AB. Application of potassium fertilisers, especially in soils with high nitrogen content, can enhance chickpea yields and retard AB (Kader et al. 1990). Tillage can be used to reduce ascospore production, since burial inhibits the teleomorph formation and maturation on infected residues (Navas-Cortes et al. 1995). Burning of chickpea stubbles in certain environments can also reduce the inoculum build up but may not be favoured because of negative effects on soil health due to loss of organic matter and essential nutrients. Chemical control Although several fungicides have proved effective in control of AB, the need for their repeated application often makes them uneconomical in regions where crop yields are low. In Australia, chickpea varieties susceptible to AB have been successfully grown by strategically applying foliar fungicides such as chlorothalonil and mancozeb several times during the growing season (Bretag et al. 2000, 2002b, 2003). Seed treatment with Calixin-M (11% tridemorph + 36% maneb) (Reddy et al. 1982), systemic methyl benzimidazole fungicides such as benomyl or thiabendazole in combination with captan (Kaiser and Hannan 1988) produced the best results in field trials. Carbendazim and thiram (1 : 1), captan, iprodione, and propiconazole (Singh and Singh 1990; Rauf et al. 1992) were all effective in control of seed-borne A. rabiei infection. Foliar application of propineb (Antracol), Bordeaux mixture, chlorothalonil, zineb, ferbam, maneb, captan, captafol, dithianon, propiconazole, penconazole, sulfur, and thiabendazole is also effective in control of AB. Application of these fungicides onto the infected crop is effective in reducing further development and secondary spread of AB (Bashir and Ilyas 1983; Bashir et al. 1987; Nene and Reddy 1987; Kaiser and Hannan 1988). Seed treatment combined with 2−3 sprays of captan, mancozeb, or chlorothalonil also effectively manages blight infection. The greatest 328 Australian Journal of Agricultural Research benefit for fungicide treatment of AB was obtained when at least one application was made before flowering (Reddy and Singh 1990). Integrated disease management Adoption of integrated disease management (IDM) practices is essential for economical and effective control of AB. Moderate levels of HPR can be combined with other cultural practices and/or application of minimum dosage of fungicides for control of AB. The locationspecific recommended IDM practices include: (a) use of pathogen-free seed, (b) seed treatment with fungicides, (c) practice of crop rotation, (d) deep ploughing of chickpea fields to bury infested debris, (e) use of disease-resistant genotypes, and ( f ) strategic application of foliar fungicides. A combination of a tolerant cv. ILC 482 and 2 sprays of chlorothalonil, one during the seedling stage and another at the early podding stage, provided the most economical field control of AB in Syria (Reddy and Singh 1990). In collaboration with the Syrian national program, ICARDA has developed an IDM package for AB management (Akem et al. 2000). The components of this package include use of tolerant cultivars adapted to early sowing, seed dressing with fungicides, single foliar application of chlorothalonil at seedling or early vegetative growth stages, and delayed sowing for lower disease impact. This package resulted in higher chickpea yields compared with the traditional spring plantings using a local variety without seed dressing or fungicide spray (ICARDA 2003). Conclusions Management of AB is essential to provide increased and stable chickpea yields throughout the world. Where possible, HPR should be emphasised over chemical control as the most environmentally friendly and economic disease control strategy. Selection of resistant sources for genetic improvement programs should be based on resistance to AB at vegetative, flowering, and podding stages, since many lines resistant in the vegetative stage can be susceptible at the podding stage. Resistance to AB in chickpea cultivars has historically been overcome by new pathotypes of A. rabiei, hence the genotypes intended for release to farmers should be selected based on multi-location multi-season field trials. Durable resistance may only be possible if an array of resistance genes is combined providing different mechanisms of resistance against all races in a single cultivar. Studies are underway to determine the genetics and allelic relationships of resistance to AB in different genotypes as an essential precursor to pyramid resistance genes. Knowledge of the variability of A. rabiei is also a prerequisite for breeding programs aimed at obtaining durable resistance to AB. Further studies on the ecology of A. rabiei and its epidemiology S. Pande et al. are required to improve the current disease management strategies. 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Ascochyta blight of chickpea ( Cicer arietinum L.): a review of biology, pathogenicity, and disease management

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